Genetically altered hybridomas, myelomas and B cells that facilitate the rapid production of monoclonal antibodies

ABSTRACT

The present invention relates to genetically altered hybridomas, myelomas and B cells. The invention also relates to utilizing genetically altered hybridomas, myelomas and B cells in methods of making monoclonal antibodies. The present invention also provides populations of hybridomas and B cells that can be utilized to make a monoclonal antibody of interest.

This application is a divisional of application Ser. No. 10/079,130,file Feb. 20, 2002 (now allowed) Now U.S. Pat. No. 7,148,040, whichclaims the benefit of U.S. provisional Patent Application No.60/270,322, filed Feb. 20, 2001 and are hereby incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to genetically altered hybridomas,myelomas and B cells. The invention also relates to utilizinggenetically altered hybridomas, myelomas and B cells in methods ofmaking monoclonal antibodies.

BACKGROUND

Major efforts in functional genomics and proteomics are creating anunprecedented demand for monoclonal antibodies to be used for proteinfunction studies. Monoclonal antibodies can be used in every stage ofresearch involving protein target discovery and characterizationincluding purification, quantification, and organ and cellularlocalization. Recent advances in proteomics are creating a need forlarge numbers of antibodies for use in high throughput studies and onprotein chips. Monoclonal antibodies have been used for decades as keyreagents in clinical diagnostics and they are emerging as an importantnew class of therapeutics agents.

Hybridoma technology is the most commonly used method for accessingmonoclonal antibodies. Monoclonal antibodies are secreted from hybridomacells, created by fusing normal antibody producing splenic B-cells withimmortal myeloma cells or other immortal cells. Hybridoma production haschanged little since its inception 26 years ago (Kohler and Milstein,1975).

A typical protocol for hybridoma generation involves: (i) immunizing ananimal (e.g., mouse, rat or rabbit) with a purified protein antigen;(ii) harvesting antibody producing B-cells, typically from the spleen;(iii) fusing B-cells with a non-secretory myeloma cell line deficientfor the enzyme hypoxanthine guanine phosphoribosyl transferase (e.g.,x63-Ag 8.653 from a BALB/c mouse strain); (iv) growing hybridoma cellsin a selection medium containing hypoxanthine, aminopterin and thymidine(HAT) and (v) screening for cells that produce the desired antibody and(vi) limit dilution cloning to obtain a homogenous cell line thatsecretes the antibody (Antczak, 1982).

Conventional hybridoma technology does not allow researchers to accesslarge numbers of antibodies to different antigens or large numbers ofantibodies to a single target antigen in an efficient manner. Hybridomacell cloning by limit dilution is perhaps the most problematic, timeconsuming and labor intensive step in generating monoclonal antibodies(O'Reilly et. al., 1998). In this step, cells are repeatedly diluted outto low cell numbers and their supernatants assayed for secretedmonoclonal antibody. Screening must be artfully performed to ensure thatdesired hybridomas are not lost. In the case of rapidly growinghybridomas, cells may die in the microtiter wells from exhaustion ofnutrients if they are not moved to larger vessels or fresh mediumquickly. Also, in typical wells containing several hybridomas,undesirable hybridomas may continuously overgrow desired hybridomas.This can cause the limit dilution step to be extended weeks or monthsand may even result in loss of important hybridomas. If the hybridomashave not grown to a reasonable size by the time of assay, they may nothave produced sufficient antibody for detection. Therefore, a time forscreening supernatants must be chosen carefully. The available “window”for initial screening is not large and usually extends over two to threedays (Antczak, 1982). Once started, the limit dilution isolation of purecell lines typically goes on for 3-4 weeks for any one hybridoma.

There is a need for more rapid methods of isolating desired hybridomacells. At least two laboratories attempted to sort normal hybridomasbased on traces of surface presentation of antibody (Parks et al., 1979;Meilhoc et al., 1989). They showed that a subset of hybridoma cells inany population presented a small but measurable number (˜20) of surfaceantibody molecules. This was enough to sort these cells when labeledwith antigen, if the antigen was coupled to highly fluorescentmicrospheres to increase the fluorescence signal. Even with these highlyfluorescent spheres the signal was only a few-fold above the background.

There is a need for a significant increase in the presentation ofsurface antibody on hybridoma cells as well as a need for a significantincrease in the percentage of hybridoma cells presenting surfaceantibody in any population to enable rapid screening. The presentinvention provides both by providing DISH (Direct Screening of HybridomaCells). The DISH technology of the present invention provides a simple,rapid and reliable selection of hybridoma cells that may be accomplishedin a matter of hours instead of weeks. DISH provides several significantimprovements over conventional hybridoma technology that allowresearchers to access much larger repertoires of antibodies in anefficient manner. Using current hybridoma protocols approximately 40,000fusions are typically prepared. A main reason more fusions are not madeis the difficulty encountered in isolating desired clones using limitdilution. DISH enables very rapid, high throughput cell selection usingfluorescence activated cell sorting (FACS) and other modalities. SinceFACS technology permits millions of cells to be sorted in a matter ofhours, the number of hybridoma fusions one can screen using DISHtechnology is orders of magnitude larger than by limit dilution.Significantly, FACS sorting allows for single cell deposition of desiredhybridomas into discrete wells. Hence, the problem of desirable, butslow growing cells being lost is eliminated using DISH. Thus, DISHreplaces current antibody screening and limit dilution procedures with arapid, high throughput, selection process. The present invention canalso be utilized to provide populations of plasma cells that surfacepresent adequate immunoglobulin to enable high throughput fluorescenceactivated cell sorting technology to be used to determine whether singleplasma cells produce immunoglobin that reacts with target antigens.

SUMMARY OF THE INVENTION

The present invention provides a population of hybridoma cells whereingreater than 15% of the cells in the population express monoclonalantibody that is bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 25% of the cells in the population expressmonoclonal antibody that is bound to the cell surface.

Further provided is a population of hybridoma cells wherein greater than50% of the cells in the population express monoclonal antibody that isbound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 75% of the cells in the population expressmonoclonal antibody that is bound to the cell surface.

The present invention also provides a hybridoma cell, wherein greaterthan twenty monoclonal antibody molecules are expressed and bound to thecell surface.

The present invention further provides a hybridoma cell, wherein greaterthan fifty monoclonal antibody molecules are expressed and bound to thecell surface.

Also provided by the present invention is a hybridoma cell, whereingreater than one hundred monoclonal antibody molecules are expressed andbound to the cell surface.

Further provided by the present invention is a hybridoma cell, whereingreater than two hundred and fifty monoclonal antibody molecules areexpressed and bound to the cell surface.

Also provided by the present invention is a hybridoma cell, whereingreater than five hundred monoclonal antibody molecules are expressedand bound to the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than twenty monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than fifty monoclonal antibody molecules are expressed and boundto the cell surface of the cells in the population that expressmonoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 25% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than twenty monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

The present invention also provides a population of hybridoma cellswherein greater than 25% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than fifty monoclonal antibody molecules are expressed and boundto the cell surface of the cells in the population that expressmonoclonal antibody.

Also provided by the present invention is a hybridoma cell, wherein fromabout 0.01% to about 10% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 0.01% to about 10% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 0.01% to about 10% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface.

Also provided by the present invention is a hybridoma cell comprising avector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ.

Further provided by the present invention is a hybridoma cell comprisinga vector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ, wherein the nucleic acid is linked to aninducible functional expression sequence.

The present invention also provides a method for making a hybridoma cellcomprising at least one surface-expressed antibody receptor selectedfrom the group consisting of Igα and Igβ comprising fusing a myelomacell comprising a vector, wherein the vector comprises a nucleic acidencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ, with a B cell to produce ahybridoma cell comprising at least one surface-expressed antibodyreceptor selected from the group consisting of Igα and Igβ.

The present invention also provides a B cell comprising a vector,wherein the vector comprises a nucleic acid encoding at least onesurface-expressed antibody receptor selected from the group consistingof Igα and Igβ.

Also provided by the present invention is a B cell comprising a vector,wherein the vector comprises a nucleic acid encoding at least onesurface-expressed antibody receptor selected from the group consistingof Igα and Igβ, wherein the vector comprises a nucleic acid encoding Igαand Igβ and wherein the vector is integrated into the genome of the Bcell.

The present invention also provides a myeloma cell comprising at leastone nucleic acid functionally encoding at least one surface-expressedantibody receptor selected from the group consisting of Igα and Igβ,wherein the nucleic acid encoding the surface-expressed antibodyreceptor is functionally linked to an inducible expression sequence.

Also provided by the present invention is a myeloma cell comprising atleast one nucleic acid functionally encoding at least one mutatedsurface-expressed antibody receptor selected from the group consistingof Igα and Igβ, wherein the nucleic acid encoding the surface-expressedantibody receptor is functionally linked to an inducible expressionsequence.

Also provided by the present invention is a method of making amonoclonal antibody of interest comprising: a) contacting a populationof hybridoma cells wherein greater than 15% of the cells in thepopulation express monoclonal antibody that is bound to the cell surfacewith an antigen linked to a detectable label, wherein the antigen bindsto the monoclonal antibody to yield a detectably labeled hybridoma cell;b) isolating the detectably labeled hybridoma cell, thus identifying ahybridoma cell that produces the monoclonal antibody of interest; and c)making the monoclonal antibody of interest from the hybridoma cell.

Also provided by the present invention is a method of making amonoclonal antibody of interest comprising: a) contacting a populationof hybridoma cells wherein greater than 15% of the cells in thepopulation express monoclonal antibody that is bound to the cell surfacewith an antigen, wherein the antigen binds to the monoclonal antibody;b) adding a detectable label to the antigen to yield a detectablylabeled hybridoma cell; c) isolating the detectably labeled hybridomacell, thus identifying a hybridoma cell that produces the monoclonalantibody of interest; and d) making the monoclonal antibody of interestfrom the hybridoma cell.

Further provided by the present invention is a method of making amonoclonal antibody of interest comprising: a) contacting a hybridomacell, wherein greater than twenty monoclonal antibody molecules areexpressed and bound to the cell surface with an antigen linked to adetectable label, wherein the antigen binds to the monoclonal antibodyto yield a detectably labeled hybridoma cell; b) isolating thedetectably labeled hybridoma cell, thus identifying a hybridoma cellthat produces the monoclonal antibody of interest; and c) making themonoclonal antibody of interest from the hybridoma cell.

Also provided by the present invention is a method of making amonoclonal antibody of interest comprising: a) contacting a hybridomacell, wherein greater than twenty monoclonal antibody molecules areexpressed and bound to the cell surface with an antigen, wherein theantigen binds to the monoclonal antibody; b) adding a detectable labelto the antigen to yield a detectably labeled hybridoma cell; c)isolating the detectably labeled hybridoma cell, thus identifying ahybridoma cell that produces the monoclonal antibody of interest; and d)making the monoclonal antibody of interest from the hybridoma cell.

Also provided by the present invention is a method of making amonoclonal antibody of interest comprising: a) contacting a B cellcomprising a vector, wherein the vector comprises a nucleic acidencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ with an antigen linked to adetectable label, wherein the antigen binds to the monoclonal antibodyto yield a detectably labeled B cell; b) isolating the detectablylabeled B cell, thus identifying a B cell that produces the monoclonalantibody of interest; and c) making the monoclonal antibody of interest.

The present invention also provides a method of making a monoclonalantibody of interest comprising: a) contacting a B cell comprising avector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ with an antigen; b) adding a detectable labelthat binds to the antigen to yield a detectably labeled B cell; c)isolating the detectably labeled B cell, thus identifying a B cell thatproduces the monoclonal antibody of interest; and d) making themonoclonal antibody of interest.

Also provided is a method of making a hybridoma cell that produces amonoclonal antibody that recognizes a selected antigen comprising: a)immunizing a mouse with the antigen; b) fusing a B cell from theimmunized mouse with a myeloma cell that comprises at least one nucleicacid functionally encoding at least one surface-expressed antibodyreceptor selected from the group consisting of Igα and Igβ to produce amonoclonal antibody producing hybridoma cell, wherein the monoclonalantibody produced by the hybridoma cell is expressed and bound to thecell surface; c) contacting the monoclonal antibody producing hybridomacell with the antigen, wherein the antigen binds to the monoclonalantibody on the cell surface to produce a detectable hybridoma cell, d)detecting the hybridoma cell and; e) isolating the hybridoma cell, thusmaking a hybridoma cell that produces a monoclonal antibody thatrecognizes a specific antigen.

The present invention also provides a transgenic animal comprising Bcells comprising a vector, wherein the vector comprises a nucleic acidencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ.

The present invention also provides a hematopoietic stem cell comprisinga vector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ.

The invention further provides a population of hybridoma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence, thefluorescence intensity of at least 10% of the cells is at least two foldgreater than a the fluorescence intensity of a population of hybridomacells that do not comprise a vector comprising a nucleic acid encodingIgα and/or Igβ.

The present invention also provides a population of plasma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence thefluorescence intensity of the population of cells is at least two foldgreater than the fluorescence intensity of a population of plasma cellsthat does not comprise a vector comprising a nucleic acid encoding Igαand/or Igβ.

The present invention also provides a population of hybridoma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence, thefluorescence intensity of at least 10% of the cells is at least two foldgreater than a the fluorescence intensity of a population of hybridomacells that do not comprise a vector comprising a nucleic acid encodingIgα and/or Igβ.

Further provided by the present invention a population of plasma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence thefluorescence intensity of at least 10% of the cells is at least two foldgreater than the fluorescence intensity of a population of plasma cellsthat do not comprise a vector comprising a nucleic acid encoding Igαand/or Igβ.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that the surface presentation of antibody such as mIgM inB-cells requires the presence of one or two membrane receptors, Igαand/or Igβ. In B-cells these receptors bind the membrane domain of themembrane form of the heavy chain (mHC, dark gray) portion of an antibodysuch as mIgM. The small immunoglobulin light chain is drawn in black. Inmost myelomas the Igα (white) receptor shown here is missing orexpressed at very low levels. In some myelomas Igα and/or Igβ may beabsent or expressed at low levels. As a result, most hybridomas cannotpresent significant amounts of mIgM on their surface.

FIG. 2 shows the Primer oligonucleotides and PCR amplification of Igαand Igβ cDNAs. (A) The sequences of the sense (S) and antisense (A)primers used to amplify cDNAs encoding Igα and Igβ (Igα sense: SEQ IDNO:5; Igα antisense: SEQ ID NO:6; Igβ sense: SEQ ID NO:7; Igβ antisense:SEQ ID NO:8). These oligos add important restriction endonucleasecloning sites and 5′ translation signals (GCCACC) to the receptorsequences. (B) PCR amplification products of the expected sizes in basepairs (bp). Molecular weight standards flank the PCR products.

FIG. 3 shows the sequence of PCR modified mouse Igα cDNA extending fromthe HindIII to EcoRI cloning sites used to construct Igα expressionplasmid p3.1Neolgα (SEQ ID NO:1). The Protein sequence is shown abovethe DNA sequence (SEQ ID NO:3). The main DNA and protein sequencelistings in GenBank for Igα have the accession numbers NM_(—)007655 andNP_(—)031681, respectively. The cDNA sequence obtained was inconsistentwith this original Igα sequence in a small region, but agrees with thedata given by Sakaguchi et al. (“B lymphocyte lineage-restrictedexpression of mb-1, a gene with CD3-like structural properties” EMBO J.7:3457-64(1988)) encoding a protein with the sequence encoding six aminoacids listed in bold.

FIG. 4 shows the sequence of PCR modified mouse Igβ cDNA extending fromthe HindIII to EcoRI cloning sites used to construct Igβ expressionplasmid p3.1ZeoIgβ (SEQ ID NO:2). Protein sequence is shown above theDNA sequence (SEQ ID NO:4). The main sequence listing in GenBank for Igβhas the accession number NM_(—)008339.

FIG. 5 shows the structure of pcDNA3.1 NeoR vector (Invitrogen, Inc.Life Sciences Division) used to express Igα receptor protein intransgenic cells.

FIG. 6 shows the structure of pcDNA3.1 Zeo vector (Invitrogen, Inc. LifeSciences Division) used to express Igβ receptor protein in transgeniccells.

FIG. 7 shows the screening of transfected HGS cell lines for Igα and Igβprotein levels on western blots. Protein samples (25 μg of total cellprotein in SDS sample buffer) were resolved on a 12% acrylamide gel,transferred to an Immobilon membrane (Millipore), reacted with rabbitpolyclonal anti mouse Igα antibody (A) or Igβ antibody (B), then withdonkey anti-rabbit horse antibody radish peroxdase conjugate, anddeveloped following published protocols (Kandasamy et al., 1999).Positions of molecular weight standards (MW Stds) are shown on the left.The range of MWs of the various modified forms of Igα are shown with abracket. Other higher bands and bands in control WT lanes appear to bebackground. Extracts from cell lines are named as follows: SC, spleencell extract; HGS1, parental hybridoma cell line producing antibodies toGS; and HGS1αβ1-5 and ECS1αβ1-3 are the receptor gene transfected celllines. References of these simplified designations to strain names inlaboratory notebooks in the Meagher laboratory at UGA are as follows:HGS1αβ1, GS-TSC; HGS1αβ2, GS-TSC-1C; HGS1αβ3, GS-TE4; HGS1αβ4, GS-T5D-L;and HGS1αβ5, GS-TSC-3D. The Igα and Igβ specific rabbit polyclonalantibodies, anti-MB-1 and SF2B, respectively, were obtained from Dr.Linda Matsuuchi (University of British Columbia, Vancouver,Canada)(Condon et al., 2000). Antibodies were prepared against syntheticpeptides to the cytoplasmic tail of Igα and the ecto-domain of Igβ.

FIG. 8 shows microscopic examination of increased surface presentationof antibody on hybridoma cells expressing transgenic Ig receptors. Allcells shown were treated with FITC labeled goat anti mouse antiserum.All plates were photographed using a 40× Zeiss lens and Hamamatsudigital camera (model C-4742-95) under the same illumination andexposure conditions. Cells treated with an unlabeled antibody haveinsignificant levels of auto-fluorescence. Cell lines shown areindicated with reference to names in lab notebooks. A & B.Non-transfected HGS1 control cells; D-H. pcDNA3.1-Igα and -Igβtransfected cells. C HGS1αβ6, SC-1C#2; D HGS1αβ7, SC-1CF2; E. HGS1αβ8,SC-1C#3; F. HGS1αβ9, SC-3DF3; G. HGS1αβ10, SC-3DF4: H. HGS1αβ11, SC-F1.(I) brightest cell enlarged from field of cells in FIG. 8A (HGS1acontrol hybridoma cells) and indicated by an arrow. (J) Brightest cellenlarged from field of cells in FIG. 8C (HGS1αβ6 receptor transfectedhybridoma cells) and indicated by an arrow.

FIG. 9 shows Igα protein levels in aliquots of HGS1 WT and HGS1αβ cellsshown surface presenting antibody in FIG. 8. Protein samples wereresolved by PAGE and Igα protein levels measured on Westerns usingrabbit antibody to mouse Igα as in FIG. 7A. (A) Igα western (B)Coomassie stained gel of duplicate samples showing relative level ofprotein loading. Stained molecular weight standards (MW Stds) are shownon the left in kilo-Daltons (kDa). The range of MWs of the variousmodified forms of Igα are shown with a bracket. Extracts from cell linesdiscussed in Meagher laboratory notebooks are named as follows: WT, HGS1non transfected control; HGS1αβ6, SC-1C#2; HGS1αβ7, SC-1CF2; HGS1αβ8,SC-1C#3; HGS1αβ9, SC-3DF3; HGS1αβ10, SC-3DF4; and HGS1αβ11, SC-F1. Celllines showing 100% surface presentation of antibody in FIG. 8 aredesignated plus +, those not presenting as minus, and HGS1 hybridoma WTcontrols with 1-5% presentation as +/−. Mobility range of Igα isoformsare shown with a bracket.

FIG. 10 shows a western blot examining Igβ expression in aliquots ofHGS1 and HGS1αβ cells shown in FIG. 8. Protein samples were resolved byPAGE and Igβ protein levels measured on Westerns using antibody to Igβas in FIG. 7B. Protein samples are identical aliquots to those shown inFIGS. 10A and 10B. Mobility range of Igβ isoforms are shown with abracket.

FIG. 11 shows quantification of surface presentation of antibody in Igαexpressing hybridomas. (A) The fluorescence intensity was compared amongHGS1 cells and transgenic cells expressing or not expressing detectablelevels of Igα receptor. Mean fluorescence intensity (MFI) was measuredfor individual cells in a microscopic field from images like those takenin FIG. 8, using OpenLab software (Improvision, Inc., Boston, Mass.).Standard errors in MFI among individual cells in each population areindicated. MFI underestimates the actual intensity differences betweencontrol and transgenic cells, because 10% of the brightest cells exceedthe dynamic range of the electronic camera and some cells are out of thefocal plane, where MFI cannot be accurately measured. n=50 cells foreach of the eight cell populations examined. (B)The frequency ofindividual cells in each population with 3 times greater intensity thanthe mean for control HGS1 cells (89 MFI).

FIG. 12 shows western blot examining Igα and Igβ protein levels intransfected myeloma cell lines. Immunodetection of (A) Igα and (B) Igβperformed as described in FIG. 7. Cell lines examined are as follows:SC, spleen cells; Sp2/0 myeloma wild-type (WT) or Sp2/0 derived linestransfected with genes encoding Igα and Igβ (αβ1, M-T4; αβ2, M-T4D; αβ3,M-T6; αβ4, M-T4-3A; αβ5, M-T3SC; αβ6, M-T1SC; and αβ7, M-T4D-6).Mobility ranges of Igα and Igβ are shown with brackets. Higher molecularweight bands are due to background activity in antibody.

FIG. 13 shows fluorescent activated cell sorting (FACS) demonstratingthat Igα expression increases antibody surface presentation eight fold.HGS1 and HGS1αβ10 cells were prepared as shown in FIG. 8 and labeledwith FITC labeled goat anti-mouse antibody. 200 μl samples of thefollowing cells were counted (Counts) by FACS using A₅₂₀ emission (FL1).The full scale on the bottom axis represents 4-logs of fluorescenceintensity. A. HGS1 WT control cells. B. HGS1αβ10 cells. C. Mixture ofHGS1 and HGS1αβ10 cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention.

Before the present methods are disclosed and described, it is to beunderstood that this invention is not limited to specific constructs,molecules and methods, as such may of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. For example, a cell canmean a single cell or more than one cell.

Hybridomas

The present invention provides a population of hybridoma cells whereingreater than 15% of the cells in the population express monoclonalantibody that is bound to the cell surface. Also provided by thisinvention is a population of hybridoma cells wherein greater than 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95% or numbers in between, of the cells in the population expressmonoclonal antibody that is bound to the cell surface.

As used herein, “hybridoma” is a cell or a cell line that is produced byfusing an antibody producing cell, e.g. a B cell, and an immortalizedcell, e.g. a myeloma cell. As used herein “B cell” means an immature Bcell, a mature naïve B cell, a mature activated B cell, a memory B cell,a B lineage lymphocyte, a plasma cell or any other B lineage cell ofhuman origin or from non-human animal sources. The hybridomas of thisinvention can be made by fusing a B cell of human origin or fromnon-human animal sources, with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, “Monoclonal Antibodies: Principles and Practice” AcademicPress, (1986) pp. 59-103).

In order to obtain the B cells for the production of a hybridoma, amouse or other appropriate host animal, is typically immunized with animmunizing agent or antigen to elicit B cells that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent or antigen. Alternatively, the B cells may be immunizedin vitro. Immortalized cell lines are usually transformed mammaliancells, particularly myeloma cells of rodent, bovine and human origin.Usually, rat or mouse myeloma cell lines are employed. The hybridomacells may be cultured in a suitable culture medium that preferablycontains one or more substances that inhibit the growth or survival ofthe unfused, immortalized cells. For example, although HAT is notnecessary for DISH, typically, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium” ), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium. Theimmortalized cell line can be sensitive to HAT medium. More preferredimmortalized cell lines are murine myeloma lines, which can be obtained,for instance, from the Salk Institute Cell Distribution Center, SanDiego, Calif. and the American Type Culture Collection (ATCC),Rockville, Md. Human myeloma and mouse-human heteromyeloma cell linesalso have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol., 133:3001 (1984) and Brodeur et al.,“Monoclonal Antibody Production Techniques and Applications” MarcelDekker, Inc., New York, (1987) pp. 51-63). For example, the followingmyeloma cell lines can be obtained from the ATCC: MOPC-31C, RPMI 8226,IM-9, MPC-11, CCL-189, HK-PEG-1, HS-Sultan, A2B5 clone 105,P3X63Ag8.653, Sp2/0-Ag14, Sp2/0-Ag14/SF, P3X63Ag8U.1, HFN 36.3 HFN 7.1,45.6.TG1.7, ARH-77, Y3-Ag 1.2.3, SJK-132-20, SJK-287-38 and SJK-237-71.

The hybridoma cells of the present invention can be assayed for surfaceexpression and the culture medium in which the hybridoma cells arecultured can be assayed for the presence of monoclonal antibodiesdirected against a desired immunogen by methods known in the art such asELISA, western blot, FACS, magnetic separation etc. The bindingspecificity of monoclonal antibodies secreted by the hybridoma cells canbe, for example, determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA). Such techniques and assays are known inthe art. The binding affinity of the monoclonal antibody can, forexample, be determined by the Scatchard analysis of Munson et al., Anal.Biochem., 107:220 (1980).

After a desired hybridoma cell is identified, either by assaying surfaceexpression or by assaying the culture medium, the selected hybridomacell can be grown by standard methods. Suitable culture media for thispurpose include, for example, Dulbecco's Modified Eagle's Medium andRPMI-1640 medium. Alternatively, the hybridoma cells may be grown invivo as ascites in a mammal.

As used herein, “a population of hybridoma cells” means a sufficientnumber of cells such that a percentage of the cells expressing antibodycan be determined. The hybridoma cells of the population can be cellsfrom a pure hybridoma cell line where all of the cells of the lineproduce only one monoclonal antibody specific for a particular antigenor a mixture of cells wherein multiple monoclonal antibodies areproduced. Thus, a population of hybridoma cells can produce more thanone monoclonal antibody such that some cells produce a monoclonalantibody that recognize one antigen and other cells in the populationproduce monoclonal antibody that recognizes a second antigen and othercells in the population produce a monoclonal antibody that recognizes athird antigen etc.

As used herein, “express” means that the monoclonal antibody can bedetected by means standard in the art such as Western blot, ELISA,immunofluorescence, hemolytic assay, fluorescence activated cell sorting(FACS) as they are currently practiced in the art.

Antibodies are typically proteins which exhibit binding specificity to aspecific antigen. Native antibodies are usually heterotetramericglycoproteins, composed of two identical light (L) chains and twoidentical heavy (H) chains. Typically, each light chain is linked to aheavy chain by one covalent disulfide bond, while the number ofdisulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain can have regularlyspaced intrachain disulfide bridges. Each heavy chain can have at oneend a variable domain (V(H) followed by a number of constant domains.Each light chain can have a variable domain at one end (V(L)) and aconstant domain at its other end; the constant domain of the light chainis aligned with the first constant domain of the heavy chain, and thelight chain variable domain is aligned with the variable domain of theheavy chain. Particular amino acid residues are believed to form aninterface between the light and heavy chain variable domains. The lightchains of antibodies from any vertebrate species can be assigned to oneof two clearly distinct types, called kappa (κ) and lambda (λ), based onthe amino acid sequences of their constant domains. Depending on theamino acid sequence of the constant domain of their heavy chains,immunoglobulins can be assigned to different classes. There currentlyare five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM,and several of these may be further divided into subclasses (isotypes),e.g., IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The presentinvention provides the presentation of all of the immunoglobulin classesvia binding to Ig α and/or Ig β. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called alpha,delta, epsilon, gamma, and mu, respectively.

The Immunoglobulin (Ig) heavy chain genes are typically complextranscription units with multiple poly(A) sites in which changes in thecleavage and polyadenylation machinery can play an important role inB-cell, stage-specific expression. Ig μ heavy chains can be expressed inpre, immature, and mature-B-cells and IgM+ plasma cells. The α, ε, and γheavy chains can be expressed in memory and IgA+, IgE+, and IgG+ plasmacells, respectively (Janeway and Travers, 1994). RNA from each of thefive classes of Ig heavy chain genes (α, δ, ε, γ, μ) can bealternatively processed to produce two types of mRNAs: one encodes thesecreted form of the Ig protein and is produced by use of thepromoter-proximal, weak Ig sec (secretory-specific) poly(A) site inplasma cells; the other mRNA encodes the membrane-bound (mb) receptorfor antigen on the surface of mature or memory B-cells and can beproduced by use of the downstream, strong Ig membrane poly(A) site [Alt,1980; Rogers, 1980; Rogers, 1981].

There can be a 2-5-fold change in the transcription rate of the Ig genesin different B-cell stages (Kelly and Perry, 1986). The site oftermination can vary in the μ (Galli et al., 1987; Guise et al., 1988;Yuan and Tucker, 1984) but not the γ and α genes (Flaspohler et al.,1995; Flaspohler and Milcarek., 1990; Lebman et al., 1992). RNAprocessing events can play the major role in determining the ratios ofthe two forms of IgG heavy chain mRNA as first shown in 1985 (Milcarekand Hall, 1985). The crucial role for RNA processing has been furthersubstantiated (See Edwalds-Gilbert and Milcarek, 1995; Edwalds-Gilbertand Milcarek, 1995; Flaspohler et al., 1995; Flaspohler and Milcarek.,1990; Genovese et al., 1991; Genovese and Milcarek, 1990; Hall andMilcarek, 1989; Kobrin et al., 1986; Lassman et al., 1992; Lassman andMilcarek, 1992; Matis et al., 1996; Milcarek et al., 1996). See also(Edwalds-Gilbert et al., 1997). Polyadenylation at the weaksecretory-specific poly(A) site, which is promoter proximal to themembrane specific poly(A) site, and splicing to the membrane-specificexons at the sub-optimal splice site, in the last secretory-specificexon, can bemutually exclusive events. It has been shown that changes inthe cleavage and polyadenylation of the precursor RNA tip the balance inplasma cells to the use of the first, weak poly(A) site.

The term “variable” is used herein to describe certain portions of thevariable domains which differ in sequence among antibodies and are usedin the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not usually evenlydistributed through the variable domains of antibodies. It is typicallyconcentrated in three segments called complementarity determiningregions (CDRs) or hypervariable regions both in the light chain and theheavy chain variable domains. The more highly conserved portions of thevariable domains are called the framework (FR). The variable domains ofnative heavy and light chains can each comprise four FR regions, largelyadopting a β-sheet configuration, connected by three CDRs, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The CDRs in each chain can be held together in closeproximity by the FR regions and, with the CDRs from the other chain,contribute to the formation of the antigen binding site of antibodies(see Kabat E. A. et al., “Sequences of Proteins of ImmunologicalInterest” National Institutes of Health, Bethesda, Md. (1987)). Theconstant domains are not typically involved directly in binding anantibody to an antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody-dependent cellular toxicity.

As used herein, “monoclonal antibody” refers to an antibody that isproduced by cells that are all derived from a single antibody-producingcell type and has a specific affinity for an antigen. Monoclonalantibodies are obtained from a substantially homogeneous population ofantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. The monoclonal antibodies secreted by thehybridoma cells of the present invention can be isolated or purifiedfrom the culture medium or ascites fluid by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography.

Once hybridomas are isolated by the present invention, the antibodycoding regions of the hybridomas can be used to makemonoclonalantibodies by recombinant DNA methods, such as those described in U.S.Pat. No. 4,816,567 or U.S. Pat. No. 6,331,415. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA may be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also may be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567) or by covalently joining to the immunoglobulin codingsequence all or part of the coding sequence for a non-immunoglobulinpolypeptide. Such a non-immunoglobulin polypeptide can be substitutedfor the constant domains of an antibody of the invention, or can besubstituted for the variable domains of one antigen-combining site of anantibody of the invention to create a chimeric bivalent antibodycomprising one antigen-combining site having specificity for one antigenand second antigen-combining site having specificity for a differentantigen.

The present invention also provides a hybridoma cell, wherein greaterthan twenty monoclonal antibody molecules are expressed and bound to thecell surface. Also provided by the present invention is a hybridomacell, wherein greater than fifty monoclonal antibody molecules areexpressed and bound to the cell surface. Further provided by the presentinvention is a hybridoma cell, wherein greater than one hundredmonoclonal antibody molecules are expressed and bound to the cellsurface. The present invention also provides a hybridoma cell, whereingreater than two hundred and fifty monoclonal antibody molecules areexpressed and bound to the cell surface. Also provided by the presentinvention is a hybridoma cell, wherein greater than five hundredmonoclonal antibody molecules are expressed and bound to the cellsurface. Numbers of antibodies in between these numbers are alsoprovided.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than twenty monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than fifty monoclonal antibody molecules are expressed and boundto the cell surface of the cells in the population that expressmonoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than one hundred monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

The present invention also provides a population of hybridoma cellswherein greater than 15% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than two hundred and fifty monoclonal antibody molecules areexpressed and bound to the cell surface of the cells in the populationthat express monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than five hundred monoclonal antibody molecules are expressedand bound to the cell surface of the cells in the population thatexpress monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 25% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than twenty monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

Also provided by the present invention is a population of hybridomacells wherein greater than 25% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than fifty monoclonal antibody molecules are expressed and boundto the cell surface of the cells in the population that expressmonoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 25% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than one hundred monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

The present invention also provides a population of hybridoma cellswherein greater than 25% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than two hundred and fifty monoclonal antibody molecules areexpressed and bound to the cell surface of the cells in the populationthat express monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 25% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than five hundred monoclonal antibody molecules are expressedand bound to the cell surface of the cells in the population thatexpress monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 50% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than twenty monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

Also provided by the present invention is a population of hybridomacells wherein greater than 50% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than fifty monoclonal antibody molecules are expressed and boundto the cell surface of the cells in the population that expressmonoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 50% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than one hundred monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

The present invention also provides a population of hybridoma cellswherein greater than 50% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than two hundred and fifty monoclonal antibody molecules areexpressed and bound to the cell surface of the cells in the populationthat express monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 50% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than five hundred monoclonal antibody molecules are expressedand bound to the cell surface of the cells in the population thatexpress monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 75% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than twenty monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

Also provided by the present invention is a population of hybridomacells wherein greater than 75% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than fifty monoclonal antibody molecules are expressed and boundto the cell surface of the cells in the population that expressmonoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 75% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than one hundred monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

The present invention also provides a population of hybridoma cellswherein greater than 75% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than two hundred and fifty monoclonal antibody molecules areexpressed and bound to the cell surface of the cells in the populationthat express monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 75% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than five hundred monoclonal antibody molecules are expressedand bound to the cell surface of the cells in the population thatexpress monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 90% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than twenty monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

Also provided by the present invention is a population of hybridomacells wherein greater than 90% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than fifty monoclonal antibody molecules are expressed and boundto the cell surface of the cells in the population that expressmonoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 90% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than one hundred monoclonal antibody molecules are expressed andbound to the cell surface of the cells in the population that expressmonoclonal antibody.

The present invention also provides a population of hybridoma cellswherein greater than 90% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than two hundred and fifty monoclonal antibody molecules areexpressed and bound to the cell surface of the cells in the populationthat express monoclonal antibody.

Further provided by the present invention is a population of hybridomacells wherein greater than 90% of the cells in the population expressmonoclonal antibody that is bound to the cell surface and whereingreater than five hundred monoclonal antibody molecules are expressedand bound to the cell surface of the cells in the population thatexpress monoclonal antibody.

Also provided by the present invention is a hybridoma cell, wherein fromabout 0.001% to about 10% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Any combinations of the above percentages of cells and number ofantibodies per cell is also provided as well as numbers in between thespecifically listed percentages of cell and number of antibodies percell.

The total amount of monoclonal antibody produced by a cell can bedetermined by any of the standard methods in the art, including, but notlimited to, Western blot, ELISA, immunofluorescence and FACS. Thesemethods are utilized to measure the amount of antibody secreted into themedium by the cell, the amount of antibody bound to the cell surface andthe amount of intracellular antibody present in the cells. One of skillin the art would know how to measure these amounts utilizing the abovetechniques or others known to the skilled artisan to obtain a totalamount of antibody produced by a cell, such that a percentage of thetotal amount of antibody that is expressed and bound to the cell surfaceis obtained.

Also provided by the present invention is a hybridoma cell, wherein fromabout 0.01% to about 10% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 10% to about 20% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 20% to about 30% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 30% to about 40% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 40% to about 50% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 50% to about 60% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 60% to about 70% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 70% to about 80% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 80% to about 90% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Also provided by the present invention is a hybridoma cell, wherein fromabout 90% to about 100% of the total amount of monoclonal antibodyproduced by the hybridoma cell is expressed and bound to the cellsurface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 0.01% to about 10% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 0.01% to about 10% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 10% to about 20% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 10% to about 20% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 20% to about 30% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 20% to about 30% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 30% to about 40% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 30% to about 40% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 40% to about 50% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 40% to about 50% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 50% to about 60% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 50% to about 60% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 60% to about 70% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 60% to about 70% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 70% to about 80% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 70% to about 80% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 80% to about 90% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 80% to about 90% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Further provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 90% to about 100% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 90% to about 100% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 0.01% to about 10% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 0.01% to about 10%of the total amount of monoclonal antibody produced by the hybridomacells on the cell surface and wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 10% to about 20% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 10% to about 20% ofthe total amount of monoclonal antibody produced by the hybridoma cellson the cell surface and wherein greater than 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 20% to about 30% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 20% to about 30% ofthe total amount of monoclonal antibody produced by the hybridoma cellson the cell surface and wherein greater than 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 30% to about 40% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 30% to about 40% ofthe total amount of monoclonal antibody produced by the hybridoma cellson the cell surface and wherein greater than 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface. Also provided bythe present invention is a population of hybridoma cells wherein greaterthan 15% of the hybridoma cells in the population express from about 40%to about 50% of the total amount of monoclonal antibody produced by thehybridoma cells on the cell surface, and wherein greater than twentymonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 40% to about 50% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan twenty monoclonal antibodies are expressed and bound to the cellsurface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 50% to about 60% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 50% to about 60% ofthe total amount of monoclonal antibody produced by the hybridoma cellson the cell surface and wherein greater than 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 60% to about 70% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 60% to about 70% ofthe total amount of monoclonal antibody produced by the hybridoma cellson the cell surface and wherein greater than 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 70% to about 80% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 70% to about 80% ofthe total amount of monoclonal antibody produced by the hybridoma cellson the cell surface and wherein greater than 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 80% to about 90% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 80% to about 90% ofthe total amount of monoclonal antibody produced by the hybridoma cellson the cell surface and wherein greater than 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 90% to about 100% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than twenty monoclonal antibodies are expressed andbound to the cell surface. Also provided by this invention is apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thehybridoma cells in the population express from about 90% to about 100%of the total amount of monoclonal antibody produced by the hybridomacells on the cell surface and wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of thepopulation are hybridoma cells wherein greater than twenty monoclonalantibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 0.01% to about 10% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 0.01% to about 10% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 10% to about 20% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 10% to about 20% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 20% to about 30% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 20% to about 30% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 30% to about 40% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 30% to about 40% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 40% to about 50% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 40% to about 50% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 50% to about 60% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 50% to about 60% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 60% to about 70% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies monoclonal antibodies are expressed and bound tothe cell surface. Also provided by this invention is a population ofhybridoma cells wherein greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the hybridoma cells inthe population express from about 60% to about 70% of the total amountof monoclonal antibody produced by the hybridoma cells on the cellsurface and wherein greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the population arehybridoma cells wherein greater than fifty, one hundred, two hundred andfifty or five hundred monoclonal antibodies are expressed and bound tothe cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 70% to about 80% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 70% to about 80% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 80% to about 90% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 80% to about 90% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by the present invention is a population of hybridomacells wherein greater than 15% of the hybridoma cells in the populationexpress from about 90% to about 100% of the total amount of monoclonalantibody produced by the hybridoma cells on the cell surface, andwherein greater than 15% of the population are hybridoma cells whereingreater than fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface. Alsoprovided by this invention is a population of hybridoma cells whereingreater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% of the hybridoma cells in the population expressfrom about 90% to about 100% of the total amount of monoclonal antibodyproduced by the hybridoma cells on the cell surface and wherein greaterthan 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, or 95% of the population are hybridoma cells wherein greaterthan fifty, one hundred, two hundred and fifty or five hundredmonoclonal antibodies are expressed and bound to the cell surface.

Also provided by this invention is a population of hybridoma cellscomprising monoclonal antibodies bound to the cell surface having abouttwo, three, four, five, six, seven, eight, nine, ten times more antibodyon the cell surface than control cells, i.e. standard hybridoma cells.

Further provided by this invention is a population of hybridoma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence, thefluorescence intensity of the population of cells is at least two foldgreater than the fluorescence intensity of a population of hybridomacells that do not comprise a vector comprising a nucleic acid encodingIgα and/or Igβ.

The invention also provides a population of hybridoma cells comprising avector comprising a nucleic acid encoding Igα and/or Igβ that expressesmonoclonal antibody bound to the cell surface, wherein when themonoclonal antibody is detected by fluorescence, the fluorescenceintensity of the population of cells is at least two fold, three fold,four fold, five fold, six fold, seven fold, eight fold, nine fold, tenfold, fifteen fold, twenty fold, thirty fold, forty fold, fifty fold,sixty fold, seventy fold, eighty fold, ninety fold, one hundred fold,two hundred and fifty fold, five hundred fold or one thousand foldgreater than the fluorescence intensity of a population of hybridomacells that do not comprise a vector comprising a nucleic acid encodingIgα and/or Igβ. The fold increase in fluorescence intensity can also beany amount in between the fold increases listed above. The fold increasein fluorescence intensity can be measured by methods standard in the artand is described herein in the Examples.

The population of hybridoma cells utilized to measure fluorescenceintensity can be between 25 and 500 cells. Therefore, the population canbe about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500 cells or any number of cells inbetween these values.

The hybridomas in the population can comprise a vector comprising anucleic acid encoding Igα.

Further provided by the present invention is a population of hybridomacells comprising a vector comprising a nucleic acid encoding Igα and/orIgβ that expresses monoclonal antibody bound to the cell surface,wherein when the monoclonal antibody is detected by fluorescence, thefluorescence intensity of at least 10% of the cells is at least two foldgreater than the fluorescence intensity of a population of hybridomacells that do not comprise a vector comprising a nucleic acid encodingIgα and/or Igβ.

The present invention also provides a population of hybridoma cellscomprising a vector comprising a nucleic acid encoding Igβ and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence, thefluorescence intensity of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100% or any percentage in between of the cells is at least two foldgreater than the fluorescence intensity of a population of hybridomacells that do not comprise a vector comprising a nucleic acid encodingIgα and/or Igβ.

The present invention also provides a population of hybridoma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence, thefluorescence intensity of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% or any percentage in between of the cells is at least twofold, three fold, five fold, six fold, seven fold, eight fold, ninefold, ten fold, twenty fold, thirty fold, forty fold, fifty fold, sixtyfold, seventy fold, eight fold, ninety fold, one hundred fold, twohundred and fifty fold, five hundred fold, one thousand fold or anyamount in between, greater than the fluorescence intensity of apopulation of hybridoma cells that do not comprise a vector comprising anucleic acid encoding Igα and/or Igβ.

Further provided by the present invention is a hybridoma cell comprisinga vector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ. Therefore, the hybridoma can comprise avector comprising a nucleic acid encoding Igα, or the hybridoma cancomprise a vector comprising a nucleic acid encoding Igβ, or thehybridoma can comprise a vector comprising a nucleic acid encoding bothIgα and a nucleic acid encoding Igβ. The nucleic acids encoding Igα orIgβ can be present in a single vector or in multiple vectors. Forexample, the hybridoma can comprise a vector comprising a nucleic acidencoding Igα and a vector comprising a nucleic acid encoding Igβ or avector comprising the nucleic acid sequences for both Igα and Igβ. Anyof the vectors can be integrated into the genome of the cell or carriedextrachromosomally to allow transient expression of Igα and/or Igβ.

As used herein, the term “nucleic acid” refers to single-or multiplestranded molecules which may be DNA or RNA, or any combination thereof,including modifications to those nucleic acids. The nucleic acid mayrepresent a coding strand or its complement, or any combination thereof.Nucleic acids may be identical in sequence to the sequences which arenaturally occurring for the Igα and Igβ receptors discussed herein ormay include alternative codons which encode the same amino acid as thatwhich is found in the naturally occurring sequence. Also contemplatedare nucleic acid sequences encoding Igα and Igβ that contain deletions,substitutions, mutations and combinations thereof as long as the nucleicacid encodes a functional receptor. These nucleic acids can also bemodified from their typical structure. Such modifications include, butare not limited to, methylated nucleic acids, the substitution of anon-bridging oxygen on the phosphate residue with either a sulfur(yielding phosphorothioate deoxynucleotides), selenium (yieldingphosphorselenoate deoxynucleotides), or methyl groups (yieldingmethylphosphonate deoxynucleotides).

The nucleic acid sequence for the Igα receptor is available from GenBAnkvia Accession No. NM_(—)007655 and the polypeptide encoded by thisnucleic acid sequence is available from GenBank via Accession NumberNP_(—)031681. The nucleic acid sequence encoding Igα that was utilizedin the Examples described herein differs from the original Igα receptoravailable from GenBAnk via Accession No. NM_(—)007655, but is consistentwith the sequence provided by Sakaguchi et al. This sequence is providedin FIG. 3.

A nucleic acid molecule encoding Igα and a nucleic acid encoding Igβ canbe isolated from the organism in which it is normally found. Forexample, a genomic DNA or cDNA library can be constructed and screenedfor the presence of the nucleic acid of interest. Methods ofconstructing and screening such libraries are well known in the art andkits for performing the construction and screening steps arecommercially available (for example, Stratagene Cloning Systems, LaJolla, Calif.). Once isolated, the nucleic acid can be directly clonedinto an appropriate vector, or if necessary, be modified to facilitatethe subsequent cloning steps. Such modification steps are routine, anexample of which is the addition of oligonucleotide linkers whichcontain restriction sites to the termini of the nucleic acid. Generalmethods are set forth in Sambrook et al ., “Molecular Cloning, aLaboratory Manual,” Cold Spring Harbor Laboratory Press (1989).

Once the nucleic acid sequence of the desired Igα and/or Igβ isobtained, the sequence encoding specific amino acids can be modified orchanged at any particular amino acid position by techniques well knownin the art. For example, PCR primers can be designed which span theamino acid position or positions and which can substitute any amino acidfor another amino acid. Then a nucleic acid can be amplified andinserted into the wild-type receptor coding sequence in order to obtainany of a number of possible combinations of amino acids at any positionof the receptors. Alternatively, one skilled in the art can introducespecific mutations at any point in a particular nucleic acid sequencethrough techniques for point mutagenesis. General methods are set forthin Smith, M. “In vitro mutagenesis” Ann. Rev. Gen., 19:423-462 (1985)and Zoller, M. J. “New molecular biology methods for proteinengineering” Curr. Opin. Struct. Biol., 1:605-610 (1991). Techniquessuch as these can be used to alter the coding sequence without alteringthe amino acid sequence that is encoded.

Another example of a method of obtaining a DNA molecule encoding Igα isto synthesize a recombinant DNA molecule which encodes Igα. A nucleicacid encoding Igβ can also be obtained in this manner. For example,oligonucleotide synthesis procedures are routine in the art andoligonucleotides coding for a particular protein region are readilyobtainable through automated DNA synthesis. A nucleic acid for onestrand of a double-stranded molecule can be synthesized and hybridizedto its complementary strand. One can design these oligonucleotides suchthat the resulting double-stranded molecule has either internalrestriction sites or appropriate 5′ or 3′ overhangs at the termini forcloning into an appropriate vector. Double-stranded molecules coding forrelatively large proteins can readily be synthesized by firstconstructing several different double-stranded molecules that code forparticular regions of the protein, followed by ligating these DNAmolecules together. For example, Cunningham, et al., “Receptor andAntibody Epitopes in Human Growth Hormone Identified by Homolog-ScanningMutagenesis,” Science, 243:1330-1336 (1989), have constructed asynthetic gene encoding the human growth hormone gene by firstconstructing overlapping and complementary synthetic oligonucleotidesand ligating these fragments together. See also, Ferretti, et al., Proc.Nat. Acad. Sci. 82:599-603 (1986), wherein synthesis of a 1057 base pairsynthetic bovine rhodopsin gene from synthetic oligonucleotides isdisclosed. By constructing a nucleic acid in this manner, one skilled inthe art can readily obtain any particular Igα or Igβ with desired aminoacids at any particular position or positions within the Igα or Igβ. Seealso, U.S. Pat. No. 5,503,995 which describes an enzyme templatereaction method of making synthetic genes. Techniques such as this areroutine in the art and are well documented. These nucleic acids orfragments of a nucleic acid encoding Igα or Igβ can then be expressed invivo or in vitro as discussed below. Similarly, nucleic acids orfragments of a nucleic acid encoding can be expressed in vivo or invitro.

Once a nucleic acid encoding Igα or a region of that nucleic acid, isconstructed, modified, or isolated, that nucleic acid can then be clonedinto an appropriate vector, which can direct the in vivo or in vitrosynthesis of that wild-type and/or modified Igα receptor protein. Also,once a nucleic acid encoding Igβ or a region of that nucleic acid, isconstructed, modified, or isolated, that nucleic acid can then be clonedinto an appropriate vector, which can direct the in vivo or in vitrosynthesis of that wild-type and/or modified Igβ receptor protein. Thevector is contemplated to have the necessary functional elements thatdirect and regulate transcription of the inserted gene, or nucleic acid.These functional elements include, but are not limited to, a promoter,regions upstream or downstream of the promoter, such as enhancers thatmay regulate the transcriptional activity of the promoter, an origin ofreplication, appropriate restriction sites to facilitate cloning ofinserts adjacent to the promoter, antibiotic resistance genes or othermarkers which can serve to select for cells containing the vector or thevector containing the insert, RNA splice junctions, a transcriptiontermination region, or any other region which may serve to facilitatethe expression of the inserted gene or hybrid gene. (See generally,Sambrook et al.). One could also transfect DNA or RNA encoding an extracopy of Igα and/or Igβ directly into the cell in the absence ofadditional functional elements, e.g. as naked DNA or RNA, as long as theIgα and/or Igβ resulted in increased expression of the receptor.

The vector comprising a nucleic acid encoding Igα and Igβ of the presentinvention can be any vector suitable for expression of a nucleic acid ina eukaryotic cell as are known in the art. For example, pcDNA3.1 NeoRvector or pcDNA 3.1 Zeo can be utilized (Invitrogen, Inc. Life SciencesDivision). Other vectors include, but are not limited to, a two vectorinducible system from Invitrogen (pIND and pVgRXR plasmids), a twovector inducible system from Clontech (pTet-ON or pTet-Off and pTRE2plasmids), single plasmids for constitutive expression from Promega (pCIor pSI plasmids), a two vector inducible system from Stratagene(pCMVLacI and pOPRSVI/MCS plasmids), single plasmid inducible systemsfrom Stratagene (pERV3 or pEGSH plasmids) and single retroviralinducible systems from Stratagene (pCFB-EGSH or pFB-ERV retroviralvectors). The vector can also be a viral vector such as an adenoviralvector, an adeno-associated viral vector, a retroviral vector, alentiviral vector, a pseudotyped retroviral vector, or a pox virusvector, such as a vaccinia virus vector.

The present invention also provides a hybridoma cell comprising avector, wherein the vector comprises a nucleic acid encoding at leastone mutated surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ. Thus, the present invention provides ahybridoma cell comprising a vector that comprises a nucleic acidencoding a mutant Igα receptor, a hybridoma cell comprising a vectorthat comprises a nucleic acid encoding a mutant Igβ receptor, and ahybridoma cell that comprises a vector that comprises both a nucleicacid encoding mutant Igα receptor and a nucleic acid encoding a mutantIgβ receptor.

The mutant Igα and Igβ receptors include non-signalling receptors withaltered cytoplasmic domains. An example of such a mutant receptor is anIgα receptor comprising mutations at amino acid residues 176, 182, 193and 204. The mutated Igα receptors of the present invention also includea mutated Igα that comprises one or more mutations selected from thegroup consisting of Y176F, Y182F, Y193F and Y204F. Further provided bythe present invention is a mutated Igα receptor that comprises adeletion of amino acid residues 176-220. Another example of a mutantsurface expressed antibody receptor is a mutated Igβ receptor comprisingmutations at amino acid residues 190 and 206. The mutated Igβ receptorsof the present invention also include a mutated Igβ receptor comprisingone or more mutations selected from the group consisting of Y190F andY206F. The mutations described herein for the Igα and the Igβ aredesigned such that the receptors retain the ability to bind antibodies,but are unable to act as signaling receptors. One of skill in the artwould know how to manipulate the nucleic acids encoding Igα and Igβ asdescribed above as well as other techniques known in the art to obtainthe mutant receptors described herein as well as other mutant receptors.

Also provided by the present invention is a hybridoma cell comprising avector, wherein the vector comprises a nucleic acid encoding at leastone chimeric surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ. As used throughout this application,“chimeric” means that the cell surface receptor can comprise a sequencederived from a receptor sequence of one species, e.g. human, and areceptor sequence derived from another species. For example, a chimericIgα can comprise a human Igα extracellular domain and a mouse Igαtransmembrane domain and mouse intracellular (cytoplasmic) domain or achimeric Igα can comprise a human Igα extracellular domain and a humantransmembrane domain and a mouse intracellular domain. Similarly, achimeric Igβ can comprise a human Igβ extracellular domain and a mouseIgβ transmembrane and mouse intracellular domain or a chimeric Igβ cancomprise a human Igβ extracellular domain and a human Igβ transmembraneand a mouse intracellular domain. Receptor sequences from other speciessuch as chicken, dog, rabbit, rat, gerbil and hamster can also beutilized to make the chimeric receptors of the present invention. Otherexamples of chimeric receptors include, but are not limited to achimeric Igα or Igβ receptor comprising a rabbit N-terminalextracellular domain, a mouse transmembrane domain and a mouseC-terminal intracellular domain; a chimeric receptor comprising achicken N-terminal domain, a mouse transmembrane domain and a mouseC-terminal domain; a chimeric receptor comprising a mouse N-terminalextracellular domain, a chicken transmembrane domain and a chickenC-terminal intracellular signaling domain; a chimeric receptorcomprising a mouse N-terminal extracellular domain, a rabbittransmembrane domain and a human C-terminal intracellular signalingdomain; a chimeric receptor comprising a mouse N-terminal extracellulardomain a human transmembrane domain and a mutant C-terminalintracellular non-signaling domain from mouse.

The chimeric receptors of this invention also include chimeric receptorscomprising a sequence derived from Igα or Igβ and another non-relatedsequence. For example, the present invention contemplates a chimeric Igαreceptor comprising an extracellular domain from a non-related protein,such as CD8 or any other protein with an extracellular domain and atransmembrane Igα domain and an intracellular Igα domain.

The present invention further provides a hybridoma cell comprising avector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ, wherein the nucleic acid is linked to aninducible functional expression sequence.

All of the sequences encoding Igα and Igβ can be functionally linked toan expression sequence. The expression sequences can include a promoter,an enhancer, a silencer and necessary information processing sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites andtranscriptional terminator sequences. The promoters utilized can beconstitutive promoters or inducible promoters.

The inducible expression systems that can be used for the compositionsand methods of the present invention include the IPTG based regulatorysystem, a tetracycline based regulatory system, CID based regulatorysystem, an ecdysone based regulatory system, and an estrogen-basedregulatory system. Burcin et al., “A Regulatory System for Target GeneExpression” Frontiers in Biosience, 3:1-7 (1998) describes these systemsin detail and is incorporated herein in its entirety for the purposes ofdescribing these inducible expression systems. Another inducible systemthat can be utilized is the cre-lox system (See Lakso “Targeted oncogeneactivation by site-specific recombination in transgenic mice.” Proc.Natl. Acad Sci USA 89: 6861-65 (1992); Orban et al., “Tissue andsite-specific DNA recombination in transgenic mice” Proc Natl Acad SciUSA 90: 6861-65 (1992); Gu et al., “Deletion of a DNA polymerase betagene segment in T cells using cell type-specific gene targeting” Science265:103-106 (1994)). The nucleic acids of the present invention can alsobe under the control of an inducible metallothionine promoter (See Coxand Maness “Neurite extension and protein tyrosine phosphorylationelicited by inducible expression of the v-src oncogene in a PC12 cellline” Exp Cell Res 195: 423-31 (1991)).

In addition to comprising vectors comprising a nucleic acid encoding atleast one surface expressed antibody receptor selected from the groupconsisting of Igα and Igβ, the hybridomas of the present invention canalso comprise a vector comprising a nucleic acid encoding U1A, an enzymeinvolved in inhibiting the expression of the secretory form ofimmunoglobulin M (See Philips et al., “Regulation of nuclear poly (A)addition controls the expression of immunoglobulin M secretory mRNA,”EMBO 22:6443-6452 (2001).

All of the hybridoma cells described in this application can be utilizedin the methods described herein to make a monoclonal antibody ofinterest.

Methods of Making Hybridomas

Also provided by the present invention is a method for making ahybridoma cell comprising at least one surface-expressed antibodyreceptor selected from the group consisting of Igα and Igβ comprisingfusing a myeloma cell comprising a vector, wherein the vector comprisesa nucleic acid encoding at least one surface-expressed antibody receptorselected from the group consisting of Igα and/or Igβ, with a B cell toproduce a hybridoma cell comprising at least one surface-expressedantibody receptor selected from the group consisting of Igα and Igβ.

In the methods of making the hybridoma cells of the present invention,the vector can integrate into the genome of the cell. The vector mayalso be carried extrachromosomally in the cell, thus allowing transientexpression of Igα and Igβ. In the methods of making the hybridomas ofthe present invention, the nucleic acids encoding Igα and Igβ can befunctionally linked to an inducible expression sequence. Inducibleexpression systems are discussed above.

The myeloma cells of the present invention can comprise at least onenucleic acid functionally encoding at least one surface-expressedantibody receptor selected from the group consisting of Igα and Igβ,wherein the nucleic acid encoding the surface-expressed antibodyreceptor is functionally linked to an inducible expression sequence. Themyeloma cells of the present invention can also comprise a nucleic acidencoding a mutated Igα receptor and/or a mutated Igβ receptor. Anexample of such a mutant receptor is an Igα receptor comprisingmutations at amino acid residues 176, 182, 193 and 204. The mutated Igαreceptors of the present invention also include a mutated Igα thatcomprises one or more mutations selected from the group consisting ofY176F, Y182F, Y193F and Y204F. Further provided by the present inventionis a myeloma cell comprising a nucleic acid encoding a mutated Igαreceptor that comprises a deletion of amino acid residues 176-220.Another example of a mutant surface expressed antibody receptor is amutated Igβ receptor comprising mutations at amino acid residues 190 and206. The mutated Igβ receptors of the present invention also include amutated Igβ receptor comprising one or more mutations selected from thegroup consisting of Y190F and Y206F. The myeloma cell of the presentinvention can be a myeloma cell comprising a vector that encodes amutant Igα receptor, a myeloma cell comprising a vector that comprises amutant Igβ receptor, or a myeloma cell that comprises a mutant Igαreceptor and a mutant Igβ receptor.

The myeloma cell utilized in the methods of the present invention canalso comprise a nucleic acid encoding a chimeric Igα receptor and/or achimeric Igβ receptor.

B Cells

The present invention also provides a B cell comprising a vector,wherein the vector comprises a nucleic acid encoding at least onesurface-expressed antibody receptor selected from the group consistingof Igα and Igβ. Therefore, the B cells of this invention can comprise avector comprising a nucleic acid encoding Igα , or a vector comprising anucleic acid encoding Igβ , or a vector comprising a nucleic acidencoding both Igα and Igβ.

The B cells of the present invention can also comprise a vectorcomprising a nucleic acid encoding a mutated Igα receptor and/or amutated Igβ receptor. An example of such a mutant receptor is an Igαreceptor comprising mutations at amino acid residues 176, 182, 193 and204. The mutated Igα receptors of the present invention also include amutated Igα that comprises one or more mutations selected from the groupconsisting of Y176F, Y182F, Y193F and Y204F. Further provided by thepresent invention is a B cell comprising a vector comprising a nucleicacid encoding a mutated Igα receptor that comprises a deletion of aminoacid residues 176-220. Another example of a mutant surface expressedantibody receptor is a mutated Igβ receptor comprising mutations atamino acid residues 190 and 206. The mutated Igβ receptors of thepresent invention also include a mutated Igβ receptor comprising one ormore mutations selected from the group consisting of Y190F and Y206F.The B cell of the present invention can be a B cell comprising a vectorthat encodes a mutant Igα receptor, a B cell comprising a vector thatencodes a mutant Igβ receptor, or a B cell that comprises a vectorencoding a mutant Igα receptor and a mutant Igβ receptor. The B cells ofthe present invention can also comprise a nucleic acid encoding achimeric Igα receptor and/or a chimeric Igβ receptor.

The present invention also provides a B cell comprising a vector,wherein the vector comprises a nucleic acid encoding Igα and Igβ,wherein the nucleic acid encoding Igα and Igβ is functionally linked toan inducible expression sequence and wherein the nucleic acid encodingIgα and Igβ is integrated into the genome of the cell. Such a B cell canbe obtained from the transgenic animals described herein.

The present invention also provides a method of making a B cellcomprising a vector, wherein the vector comprises a nucleic acidencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ comprising the steps of transfectinga B cell with a vector comprising at least one nucleic acid functionallyencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ, wherein the nucleic acid encodingthe surface-expressed antibody receptor is functionally linked to anexpression sequence. In the methods of making the B cells of the presentinvention, the nucleic acids encoding Igα and Igβ can be functionallylinked to an inducible expression sequence. In the methods of making theB cells of the present invention, the vector comprising at least onenucleic acid functionally encoding at least one surface-expressedantibody receptor selected from the group consisting of Igα and Igβ canbe integrated into the genome of the B cell. Alternatively, the vectordoes not integrate into the genome of the cell and the vector is carriedextrachromosomally to allow transient expression of Igα and/or Igβ.

The vectors of the present invention can be transfected into cells usingany technique known in the art. For example, lipofectamine transfection,microinjection, electroporation, liposomal delivery and particle gunbombardment can all be utilized to effect vector delivery to cells.

In order to transfect B cells removed from an animal's spleen, B cellscan be propagated for 24-48 hours in culture so that they divide. Aretroviral vector comprising a nucleic acid encoding comprising at leastone nucleic acid functionally encoding at least one surface-expressedantibody receptor selected from the group consisting of Igα and Igβ canthen be transfected into the cells. In order to promote proliferation,cytokines can be added to the culture. Cytokines that can be utilized inthese methods include, but are not limited to, IL-2, IL-4, IL-5, IL-6,IFN-γ, and/or TGF-β. B cells that produce antibodies bound to their cellsurface can then be detected by methods known in the art and describedherein, such as FACS, cell panning, ELISA etc. Alternatively, a viralvector such as an adenoviral vector, a lentiviral vector, anadeno-associated vector, a vaccinia virus vector, a pseudotypedretroviral vector can be utilized to transfect B cells. One of skill inthe art would know how to test B cells for their ability to betransfected by a vector and select the vector that is most suitable forthe introduction of nucleic acids into these cells. One of skill in theart can also engineer the B cells of the present invention to produce acell surface receptor that would be recognized by a particular vector.For example, the B cells of the present invention could be engineeredsuch that a cell-surface receptor for adenovirus is present on the cellsurface of the B cells in order to facilitate entry of the adenoviralvector into the B cells. Alternatively, a viral vector comprising aligand that binds to a receptor (other than Igα and Igβ that has beenintroduced into the B cell can also be utilized to effect vectortransfer. The presence of these cell surface receptors can be controlledby an inducible expression system described herein or elsewhere in theart, such that after transfection, expression of the cell surfacereceptor necessary for viral entry is no longer induced and thus thereceptor is no longer expressed on the cell surface of the B cell. TheseB cells can then be fused to an immortalized cell, such as a naturallyoccurring myeloma or a genetically altered myeloma cell, such as thoseprovided herein to make a hybridoma cell line expressing a monoclonalantibody of interest. Alternatively the amino acid sequence of theantibody or desired portion thereof, such as a variable region made bysuch B cell, or the nucleic acid (cDNA) that codes for such antibody orportion thereof, may be determined or isolated. Such DNA sequence maythen be incorporated into a vector which directs the expression andsecretion of such antibody and such vector transfected into a host cell,such as a myeloma or other appropriate immortal cell. Techniques fordetermining transfecting and expressing such antibody sequences aredescribed in U.S. Pat. No. 5,627,052 and U.S. Pat. No. 6,331,415.

The B cells of the present invention can further comprise a detectablelabel.

The present invention also provides an immortalized B cell made bytransfecting the B cell with a nucleic acid encoding telomerase. (SeeBunk “Immortalizing Human Cells” , The Scientist 14:19 (2000)).Immortalized B cells can also be made be inactivating pRB/p 16 (INK4a)in addition to enhanced telomerase expression (See Kiyono et al., 1998;Dickson et al., 2000). Furthermore, immortalized cells can be made byoverexpressing c-myc and simian virus 40 large T antigen (Greenberg etal., 1999; Kim et al., 2001). Immortalized B cells can also be made byoverexpressing Cyclin D1 and inactivating p 53 (See Opitz et al., 2001)or by overexpressing SV40 large T antigen alone (Russo et al., 1998).Other methods of immortalizing B cells include overexpressing ras genesand overexpressing human papillomavirus 16E6 and E7 genes (See Coursenet al., 1997). Another combination of genes that can be utilized ishTERT, sv40 large T oncoprotein and an onco-allele of H-ras.

For some applications it may be desirable to generate B cells that arecapable of expressing one or more Ig receptors and are also immortal.One means of achieving this is to use embryos derived from an animalthat is transgenic for one or more immortalizing genes. One such animalis an IMMORTOMOUSE® mouse, commercially available through Charles RiverLaboratories. Such mice have a temperature sensitive SV40 T antigen genein most cells. Those of ordinary skill in the art will recognize thatimmortal B cells may also be obtained by using transgenic animals thatcarry additional genes known to immortalize cells as described abovesuch as hTERT or H-ras.

In addition to comprising vectors comprising a nucleic acid encoding atleast one surface expressed antibody receptor selected from the groupconsisting of Igα and Igβ, the B cells of the present invention can alsocomprise a vector comprising a nucleic acid encoding U1A, an enzymeinvolved in inhibiting the expression of the secretory form ofimmunoglobulin M (See Philips et al., “Regulation of nuclear poly (A)addition controls the expression of immunoglobulin M secretory mRNA ” ,EMBO 22:6443-6452 (2001).

All of the B cells comprising vectors described herein can be fused to amyeloma cell or other immortal cell line to make a hybridoma cell. Theresulting hybridoma cell can be utilized in the methods of making amonoclonal antibody of interest described herein.

Plasma Cells

There are approximately 10⁸ B cells in a typical mouse spleen. About 99%of such B cells surface present antibody. However 1%, or 10⁶ of suchcells are plasma cells and typically surface present only trace amountsof antibody. Plasma cells are known to produce immunoglobin that ishighly specific and of strong affinity for particular target antigens.This invention provides populations of plasma cells that surface presentadequate immunoglobin to enable high throughput fluorescence activatedcell sorting technology to be used to determine whether single cellsproduce immunoglobin that react with target antigens. Such plasma cellsmay be obtained from any animal such as the transgenic animals providedherein and can produce fully human immunoglobin, if isolated from atransgenic animal that expresses a nucleic acid coding for humanantibodies in its B cells.

As stated above, plasma cells make very specific, high affinityantibodies to target antigens. Therefore, it is desirable to isolateplasma cells from among a larger population of B cells prior to sortingthe plasma cells to identify cells that produce desired antibodies. Themarker SYNDECAN-1 is expressed to a higher degree on plasma cells thanon other B cells. In addition, plasma cells do not express IGD or B220,whereas other B cells do express both markers. Commercial antibodies forSYNDECAN, IGD and B220 are available and the three markers may be usedby those of ordinary skill in the art to segregate plasma cells fromamong B cell populations by methods known in the art. Plasma cells mayalso be separated from other cells by density-based centrifugation wherethe fraction containing plasma cells is collected using an elutriator.Alternatively, a purified plasma cell population may be achieved usingseparation/purification columns such as those that utilizing magneticbeads.

The present invention provides a population of plasma cells whereingreater than 5% of the cells in the population express monoclonalantibody that is bound to the cell surface. Also provided by thisinvention is a population of plasma cells wherein greater than 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, or 95% of the cells in the population express monoclonal antibodythat is bound to the cell surface.

The present invention also provides a plasma cell, wherein greater thantwenty monoclonal antibody molecules are expressed and bound to the cellsurface. Also provided by the present invention is a plasma cell,wherein greater than fifty monoclonal antibody molecules are expressedand bound to the cell surface. Further provided by the present inventionis a plasma cell, wherein greater than one hundred monoclonal antibodymolecules are expressed and bound to the cell surface. The presentinvention also provides a plasma cell, wherein greater than two hundredand fifty monoclonal antibody molecules are expressed and bound to thecell surface. Also provided by the present invention is a plasma cell,wherein greater than five hundred monoclonal antibody molecules areexpressed and bound to the cell surface.

Further provided by this invention is a population of plasma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence, thefluorescence intensity of the population of cells is at least two foldgreater than the fluorescence intensity of a population of plasma cellsthat do not comprise a vector comprising a nucleic acid encoding Igαand/or Igβ.

The invention also provides a population of plasma cells comprising avector comprising a nucleic acid encoding Igα and/or Igβ that expressesmonoclonal antibody bound to the cell surface, wherein when themonoclonal antibody is detected by fluorescence, the fluorescenceintensity of the population of cells is at least two fold, three fold,four fold, five fold, six fold, seven fold, eight fold, nine fold, tenfold, fifteen fold, twenty fold, thirty fold, forty fold, fifty fold,sixty fold, seventy fold, eighty fold, ninety fold, one hundred fold,two hundred and fifty fold, five hundred fold, one hundred fold, twohundred and fifty fold, five hundred fold or one thousand fold greaterthan the fluorescence intensity of a population of plasma cells that donot comprise a vector comprising a nucleic acid encoding Igα and/or Igβ.The fold increase in fluorescence intensity can also be any amount inbetween the fold increases listed above. The fold increase influorescence intensity can be measured by methods standard in the artand is described herein in the Examples.

The population of plasma cells utilized to measure fluorescenceintensity can be between 25 and 500 cells. Therefore, the population canbe about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500 cells or any number of cells inbetween these values.

The plasma cells in the population can comprise a vector comprising anucleic acid encoding Igα.

Further provided by the present invention is a population of plasmacells comprising a vector comprising a nucleic acid encoding Igα and/orIgβ that expresses monoclonal antibody bound to the cell surface,wherein when the monoclonal antibody is detected by fluorescence, thefluorescence intensity of at least 10% of the cells is at least two foldgreater than the fluorescence intensity of a population of plasma cellsthat do not comprise a vector comprising a nucleic acid encoding Igαand/or Igβ.

The present invention also provides a population of plasma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence, thefluorescence intensity of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, 100% or any percentage in between of the cells is at least two foldgreater than the fluorescence intensity of a population of plasma cellsthat do not comprise a vector comprising a nucleic acid encoding Igαand/or Igβ.

The present invention also provides a population of plasma cellscomprising a vector comprising a nucleic acid encoding Igα and/or Igβthat expresses monoclonal antibody bound to the cell surface, whereinwhen the monoclonal antibody is detected by fluorescence, thefluorescence intensity of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 100% or any percentage in between of the cells is at least twofold, three fold, five fold, six fold, seven fold, eight fold, ninefold, ten fold, twenty fold, thirty fold, forty fold, fifty fold, sixtyfold, seventy fold, eight fold, ninety fold, one hundred fold, twohundred and fifty fold, five hundred fold, one thousand fold or anyamount in between, greater than the fluorescence intensity of apopulation of plasma cells that do not comprise a vector comprising anucleic acid encoding Igα and/or Igβ.

All of the populations of plasma cells described herein can be utilizedin the methods of making antibodies provide by the present inventionsuch that the plasma cells of the present invention can be contactedwith an antigen/antigens in order to identify monoclonal antibodyproducing cells which can be isolated and subsequently produced.

Myeloma Cells

The present invention also provides a myeloma cell that comprises atleast one nucleic acid functionally encoding at least onesurface-expressed antibody receptor selected from the group consistingof Igα and Igβ, wherein the nucleic acid encoding the surface-expressedantibody receptor is functionally linked to an inducible expressionsequence.

Further provided by the present invention is a myeloma cell thatcomprises at least one nucleic acid functionally encoding at least onemutated surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ, wherein the nucleic acid encoding thesurface-expressed antibody receptor is functionally linked to aninducible expression sequence.

Also provided by the present invention is a myeloma cell that comprisesa nucleic acid functionally encoding a mutated Igα receptor having adeletion of amino acid residues 176-220, wherein the nucleic acidencoding the surface-expressed antibody receptor is functionally linkedto an inducible expression sequence.

Further provided by this invention is a myeloma cell that comprises anucleic acid functionally encoding a mutated Igα receptor having one ormore mutations selected from the group consisting of: Y176F, Y182F,Y193F, Y204F, wherein the nucleic acid encoding the surface-expressedantibody receptor is functionally linked to an inducible expressionsequence.

The present invention further provides a myeloma cell that comprises anucleic acid functionally encoding a mutated Igβ receptor having one ormore mutations selected from the group consisting of: Y190F and Y206F,wherein the nucleic acid encoding the surface-expressed antibodyreceptor is functionally linked to an inducible expression sequence.

Any of the myeloma cells comprising vectors provided by the presentinvention can be fused to a B cell, including a B cell comprising avector of the present invention, to make a hybridoma cell. The resultinghybridoma cell can then be used in the methods of making monoclonalantibodies described herein.

The present invention also provides a method of making a myeloma cellthat comprises at least one nucleic acid functionally encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ, wherein the nucleic acid encoding thesurface-expressed antibody receptor is functionally linked to aninducible expression sequence comprising the steps of transfecting amyeloma cell with at least one nucleic acid functionally encoding atleast one surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ, wherein the nucleic acid encoding thesurface-expressed antibody receptor is functionally linked to aninducible expression sequence.

The present invention also provides a method of making a myeloma cellthat comprises at least one nucleic acid functionally encoding at leastone mutated surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ, wherein the nucleic acid encoding thesurface-expressed antibody receptor is functionally linked to aninducible expression sequence comprising transfecting a myeloma cellwith at least one nucleic acid functionally encoding at least onemutated surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ, wherein the nucleic acid encoding thesurface-expressed antibody receptor is functionally linked to aninducible expression sequence.

Also provided by the present invention is a method of making a myelomacell that comprises a nucleic acid functionally encoding a mutated Igβreceptor having one or more mutations selected from the group consistingof: Y176F, Y182F, Y193F, Y204F, wherein the nucleic acid encoding thesurface-expressed antibody receptor is functionally linked to aninducible expression sequence comprising transfecting a myeloma cellwith a nucleic acid functionally encoding a mutated Igα receptor havingone or more mutations selected from the group consisting of: Y176F,Y182F, Y193F, Y204F, wherein the nucleic acid encoding thesurface-expressed antibody receptor is functionally linked to aninducible expression sequence.

The present invention provides a method of making a myeloma cell thatcomprises a nucleic acid functionally encoding a mutated Igβ receptorhaving one or more mutations selected from the group consisting of:Y190F and Y206F, wherein the nucleic acid encoding the surface-expressedantibody receptor is functionally linked to an inducible expressionsequence comprising transfecting a myeloma cell with a nucleic acidfunctionally encoding a mutated Igβ receptor having one or moremutations selected from the group consisting of: Y190F and Y206F,wherein the nucleic acid encoding the surface-expressed antibodyreceptor is functionally linked to an inducible expression sequence.

The present invention provides a method of making a myeloma cell thatcomprises a nucleic acid functionally encoding a mutated Igβ receptorhaving a deletion of amino acid residues 176-220, wherein the nucleicacid encoding the surface-expressed antibody receptor is functionallylinked to an inducible expression sequence comprising transfecting amyeloma cell with a nucleic acid functionally encoding a mutated Igβreceptor having a deletion of amino acid residues 176-220, wherein thenucleic acid encoding the surface-expressed antibody receptor isfunctionally linked to an inducible expression sequence.

In addition to comprising vectors comprising a nucleic acid encoding atleast one surface expressed antibody receptor selected from the groupconsisting of Igα and Igβ, the myeloma cells of the present inventioncan also comprise a vector comprising a nucleic acid encoding U1A, anenzyme involved in inhibiting the expression of the secretory form ofimmunoglobulin M (See Philips et al., “Regulation of nuclear poly (A)addition controls the expression of immunoglobulin M secretory mRNA” ,EMBO 22:6443-6452 (2001).

The present invention also provides a method of screening myeloma cellsor other immortal cells for the presence of Igα and Igβ on theirsurface. If a myeloma cell or am immortal cell naturally expressing Igαand Igβ is identified, this cell can be fused to B cells to producehybridoma cells expressing monoclonal antibodies on their cell surface.If a myeloma cell or an immortal cell naturally expressing Igα isidentified, this cell can be fused to the B cells of the presentinvention to produce hybridoma cells expressing monoclonal antibodies ontheir cell surface. Alternatively, this cell can be transfected with avector comprising a nucleic acid encoding Igβ and then fused to a B cellin order to produce hybridoma cells expressing monoclonal antibodies ontheir cell surface. If a myeloma cell or an immortal cell naturallyexpressing Igβ is identified, this cell can be fused to the B cells ofthe present invention to produce hybridoma cells expressing monoclonalantibodies on their cell surface. Alternatively, this cell can betransfected with a vector comprising a nucleic acid encoding Igα andthen fused to a B cell in order to produce hybridoma cells expressingmonoclonal antibodies on their cell surface.

Myeloma cells can also be screened to determine which of the myelomacells is a suitable fusion partner for making hybridomas. One of skillin the art would know how to test myelomas for desirable fusioncharacteristics either before or after screening for the presence of Igαand/or Igβ in order to determine which ones are best suited for fusionwith B cells. Alternatively, once a myeloma cell or immortalized cell isdeemed to be suitable for fusion, this myeloma cell can be transfectedwith Igα and/or Igβ prior to fusion with a B cell. In addition, sinceHAT selection is not required, the investigator's choice of the cell tobe used as a fusion partner for B cells in a given protocol is greatlyexpanded. Using DISH, myelomas or other candidate fusion partners can beidentified that are more cell sparing or offer other advantages overstandard myelomas in use today.

Methods of Making Monoclonal Antibodies

The present invention provides method of making a monoclonal antibody ofinterest comprising: a) contacting a population of hybridoma cellswherein greater than 15% of the cells in the population expressmonoclonal antibody that is bound to the cell surface with an antigenlinked to a detectable label, wherein the antigen binds to themonoclonal antibody to yield a detectably labeled hybridoma cell; b)isolating the detectably labeled hybridoma cell, thus identifying ahybridoma cell that produces the monoclonal antibody of interest; c)making the monoclonal antibody of interest from the hybridoma cell.

Also provided is a method of making a monoclonal antibody of interestcomprising: a) contacting a population of hybridoma cells whereingreater than 15% of the cells in the population express monoclonalantibody that is bound to the cell surface with an antigen, wherein theantigen binds to the monoclonal antibody; b) adding a detectable labelto the antigen to yield a detectably labeled hybridoma cell; c)isolating the detectably labeled hybridoma cell, thus identifying ahybridoma cell that produces the monoclonal antibody of interest; d)making the monoclonal antibody of interest from the hybridoma cell.

In the methods of making a monoclonal antibody described herein,conditions whereby an antigen/antibody complex can form as well asassays for the detection of the formation of an antigen/antibody complexand quantitation of the detected protein are standard in the art. Suchassays can include, but are not limited to, Western blotting,immunoprecipitation, immunofluorescence, immunocytochemistry,immunohistochemistry, fluorescence activated cell sorting (FACS),fluorescence in situ hybridization (FISH), immunomagnetic assays, ELISA,ELISPOT (Coligan et al., eds., Current Protocols in Immunology, Wiley,New York (1995)), agglutination assays, flocculation assays, cellpanning, magnetic separation etc., as are well known to those of skillin the art.

The antigen of this invention can be bound to a substrate (e.g., beads,tubes, slides, plates, nitrocellulose sheets, etc.) or conjugated with adetectable label (moiety) or both bound and conjugated. The detectablemoieties contemplated for the present invention can include, but are notlimited to, an immunofluorescence moiety (e.g., fluorescein, rhodamine),a radioactive moiety (e.g., ³²P, ¹²⁵I, ³⁵S), an enzyme moiety (e.g.,horseradish peroxidase, alkaline phosphatase), a colloidal gold moiety,a dye and a biotin moiety. Such conjugation techniques are standard inthe art (see, e.g., Harlow and Lane, “Antibodies, A Laboratory Manual”Cold Spring Harbor Publications, New York, (1988); Yang et al., Nature382: 319-324 (1996)). Labels can be coupled either directly orindirectly to the antigens. One example of indirect coupling is by useof a spacer moiety. These spacer moieties, in turn, can be eitherinsoluble or soluble (Diener et al., Science 231: 148 (1986)).

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,fluorescent compounds, chemiluminescent compounds, microspheres dyes andbioluminescent compounds. Furthermore, the binding of these labels tothe antigens required for practice of the invention can be done usingstandard techniques common to those of ordinary skill in the art.

Since the populations of hybridomas described herein can produce morethan one monoclonal antibody, the present invention provides for methodsof making monoclonal antibodies, wherein the population of cells iscontacted with more than one antigen. Once each antigen binds amonoclonal antibody of interest, each one can be detected by a separatelabel, thus identifying more than one monoclonal antibody of interest ina population of cells. For example, the population can be contacted withthree antigens, wherein each antigen is labeled either directly orindirectly with a different fluorescent label. The monoclonal antibodyproducing cells can be detected and the three different monoclonalantibody producing cells can be distinguished based on the differencesin fluorescence associated with the different labels. Therefore, thepresent invention allows the isolation and production of more than onemonoclonal antibody from a population of cells. This same approach canbe applied to the isolation and production of multiple monoclonalantibodies from the B cells of this invention, including the isolationand production of monoclonal antibodies from the plasma cells of thisinvention.

In the above methods of making a monoclonal antibody of interest, apopulation of hybridoma cells wherein greater than 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% can becontacted with the antigen.

A method of making a monoclonal antibody of interest comprising: a)contacting a hybridoma cell, wherein greater than twenty monoclonalantibody molecules are expressed and bound to the cell surface with anantigen linked to a detectable label, wherein the antigen binds to themonoclonal antibody to yield a detectably labeled hybridoma cell; b)isolating the detectably labeled hybridoma cell, thus identifying ahybridoma cell that produces the monoclonal antibody of interest; c)making the monoclonal antibody of interest from the hybridoma cell.

A method of making a monoclonal antibody of interest comprising: a)contacting a hybridoma cell, wherein greater than twenty monoclonalantibody molecules are expressed and bound to the cell surface with anantigen, wherein the antigen binds to the monoclonal antibody; b) addinga detectable label to the antigen to yield a detectably labeledhybridoma cell; c) isolating the detectably labeled hybridoma cell, thusidentifying a hybridoma cell that produces the monoclonal antibody ofinterest; d) making the monoclonal antibody of interest from thehybridoma cell.

In the above methods of making a monoclonal antibody of interest, ahybridoma cell wherein greater than fifty, one hundred, two hundred andfifty or five hundred monoclonal antibody molecules are expressed andbound to the cell surface can be contacted with the antigen.

In all of the methods of making a monoclonal antibody of interest, thehybridoma cells can comprise a vector comprising a nucleic acid encodingat least one surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ. The nucleic acid encoding at least onesurface-expressed antibody receptor selected from the group consistingof Igα and Igβ can be a mutated Igα and/or mutated Igβ, a chimeric Igαand/or chimeric Igβ as described above.

The present invention also provides a method of making a monoclonalantibody of interest comprising: a) contacting a B cell comprising avector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ with an antigen linked to a detectable label,wherein the antigen binds to the monoclonal antibody to yield adetectably labeled B cell; b) isolating the detectably labeled B cell,thus identifying a B cell that produces the monoclonal antibody ofinterest; and c) making the monoclonal antibody of interest.

The present invention also provides a method of making a monoclonalantibody of interest comprising: a) contacting a B cell comprising avector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ with an antigen; b) add a detectable labelthat binds to the antigen to yield a detectably labeled B cell; c)isolating the detectably labeled B cell, thus identifying a B cell thatproduces the monoclonal antibody of interest; and d) making themonoclonal antibody of interest.

The invention further provides a method of making a monoclonal antibodyof interest comprising: a) contacting a B cell comprising a vector,wherein the vector comprises a nucleic acid encoding at least onesurface-expressed antibody receptor selected from the group consistingof Igα and Igβ with an antigen linked to a detectable label, wherein theantigen binds to the monoclonal antibody to yield a detectably labeled Bcell; b) isolating the detectably labeled B cell, thus identifying a Bcell that produces the monoclonal antibody of interest; c) determiningthe amino acid sequence of the variable region of the monoclonalantibody; and d) making the monoclonal antibody of interest. The aminoacid sequence of the variable region of the monoclonal antibody can bedetermined by obtaining RNA from an antibody producing cell, such as a Bcell, constructing a cDNA, amplifying the cDNA by utilizing primerscorresponding to a DNA sequence in the variable region of theimmunoglobulin chain, determining the nucleotide sequence andtranslating the nucleotide sequence in order to obtain an amino acidsequence for the variable region of the monoclonal antibody. For thepurposes of determining the amino acid sequence of the variable regionof a monoclonal antibody, please see, U.S. Pat. No. 5,627,052 which ishereby incorporated in its entirety by this reference.

The invention further provides a method of making a monoclonal antibodyof interest comprising: a) contacting a B cell comprising a vector,wherein the vector comprises a nucleic acid encoding at least onesurface-expressed antibody receptor selected from the group consistingof Igα and Igβ with an antigen linked to a detectable label, wherein theantigen binds to the monoclonal antibody to yield a detectably labeled Bcell; b) isolating the detectably labeled B cell, thus identifying a Bcell that produces the monoclonal antibody of interest; c) obtaining anucleic acid encoding the variable region of the monoclonal antibody andd) making the monoclonal antibody of interest. A nucleic acid encodingthe variable region of the monoclonal antibody can be obtained byisolating DNA from an antibody producing cell, such as a B cell. OnceDNA is isolated, the DNA sequences encoding the rearranged variableregions, including the complementarity determining regions are amplifiedby PCR and the resulting amplification product sequenced. For thepurposes of obtaining a nucleic acid encoding the variable region of amonoclonal antibody, please see U.S. Pat. No. 5,627,052 which is herebyincorporated in its entirety by this reference.

In all of the methods of making a monoclonal antibody of interest from Bcells, the B cells can comprise a vector comprising a nucleic acidencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ. The nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ can be a mutated Igα and/or mutated Igβ, achimeric Igα and/or chimeric Igβ.

The present invention also provides a method of making a hybridoma cellthat produces a monoclonal antibody that recognizes a selected antigencomprising: a) immunizing a mouse with the antigen; b) fusing a B cellfrom the immunized mouse with a myeloma cell that comprises at least onenucleic acid functionally encoding at least one surface-expressedantibody receptor selected from the group consisting of Igα and Igβ toproduce a monoclonal antibody producing hybridoma cell, wherein themonoclonal antibody produced by the hybridoma cell is expressed andbound to the cell surface; c) contacting the monoclonal antibodyproducing hybridoma cell with the antigen, wherein the antigen binds tothe monoclonal antibody on the cell surface to produce a detectablehybridoma cell, and d) isolating the detectable hybridoma cell, thusmaking a hybridoma cell that produces a monoclonal antibody thatrecognizes a specific antigen. In this method, the antigen can bedirectly labeled to yield a detectably labeled hybridoma cell.

As used herein, an “antigen” can be a peptide, a polypeptide, arecombinant polypeptide, a carbohydrate, a nucleic acid, a lipid, afragment of a polypeptide, such as a C-terminal fragment or anN-terminal fragment, an organic compound, a synthetic compound, anaturally occurring compound derived from bacterial, plant, animal,protist or fungal source. The antigen can also comprise the binding siteof a cell surface receptor such that monoclonal antibodies against thatparticular site can be made to target a cell surface receptor.

A method of making a hybridoma cell that produces a monoclonal antibodythat recognizes a selected antigen comprising: a) contacting a B cellcomprising a vector, wherein the vector comprises a nucleic acidencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ with an antigen wherein the antigenbinds to the monoclonal antibody to yield a detectable B cell; b)isolating the detectable B cell, thus identifying a B cell that producesthe monoclonal antibody of interest and; c) fusing the B cell thatproduces the monoclonal antibody of interest to a myeloma cell toproduce a hybridoma cell that produces a monoclonal antibody thatrecognizes a selected antigen. In this method, the antigen can bedirectly labeled to yield a detectably labeled B cell.

Also provided by this invention is a transgenic animal comprising Bcells comprising a vector, wherein the vector comprises a nucleic acidencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ functionally linked to expressionsequences, including but not limited to a promoter, intronic sequencesand poly-adenylation signal sequences. The B cells comprising a vectorcan comprise at least one mutated surface-expressed antibody receptorselected from the group consisting of Igα and Igβ. The B cellscomprising a vector can comprise at least one chimeric surface-expressedantibody receptor selected from the group consisting of Igα and Igβ. TheB cells comprising a vector can comprise a mutated Igα receptorcomprising one or more mutations selected from the group consisting of:Y176F, Y182F, Y193F, Y204F. The B cells comprising a vector can comprisea mutated Igβ receptor comprising one or more mutations selected fromthe group consisting of: Y190F and Y206F.

The transgenic animals of this invention can be made by methods known inthe art. For the purposes of generating a transgenic animal, screeningthe transgenic animal for the presence of a transgene and othermethodology regarding transgenic animals, please see U.S. Pat. No.6,111,166 which is incorporated by this reference in its entirety. Forexample, the transgenic animals of this invention can be made by a)injecting a transgene comprising a nucleic acid encoding Igαfunctionally linked to an expression sequence and/or a transgenecomprising a nucleic acid encoding Igβ functionally linked to anexpression sequence into an embryo and b) allowing the embryo to developinto an animal. This can further comprise crossing the animal with asecond animal to produce a third animal. B cells comprising a transgene,wherein the transgene comprises a nucleic acid encoding at least onesurface-expressed antibody receptor selected from the group consistingof Igα and Igβ can be isolated from the transgenic animal of thisinvention. The transgenic animals of the present invention include, butare not limited to, mouse, rat, rabbit, guinea pig.

In the transgenic animals of the present invention, the transgene can beexpressed in immature B cells, mature naive B cells, mature activated Bcells, memory B cells, B lineage lymphocytes and/or plasma cells.Therefore, the expression sequences can be selected such that expressionof the transgene is directed to B cells, but not exclusively to B cells.The expression sequence can direct expression to one, more than one orall of the following types of B cells: immature B cells, mature naive Bcells, mature activated B cells, memory B cells, B lineage lymphocytesand plasma cells.

In the transgenic animals of the present invention, expression of thetransgene can be controlled by an inducible promoter. The transgenicanimal of this invention can utilize an inducible expression system suchas the cre-lox, metallothionine, or tetracycline-regulatedtransactivator system. Using the example of the cre-lox system, thegenes of interest (Igα and Igβ) are inserted onto a plasmid or suitableviral vector containing a stop codon flanked by locus of crossing over(loxP) sites which comprise two 13 base pair inverted repeats separatedby an 8 base pair spacer region. This cassette is under control of aspecific promoter such as the immunoglobulin kappa, immunoglobulinlambda, CD19, CD45R/B220, CD81 (TAPA-1), or CD138 (syndecan-1) promoter.The genes of interest are inserted in the plasmid on the opposite sideof the loxP-stop-loxP region from the cell specific promoter. In anotherplasmid, cre-recombinase is inserted next to a promoter whose expressionmay be controlled (proI). Each plasmid is micro-injected into thepronuclei of separate embryos and the embryos implanted into apseudopregnant female. Additionally, the plasmids may be used totransform embryonic stem cells from a suitable animal. The latter willthereafter be combined with blastocysts from the same or similarnon-human animal and re-implanted into pseudopregnant foster mothers togenerate chimeric animals comprising the plasmid comprising thetransgene. Further methods of generating transgenic animals well knownin the art, such as lipofectin or viral transfection of embryonic stemcells or pre-implantation embryos, may also be used. Alternatively, micebearing a proI-cre transgene may include already established mice suchas the interferon inducible ‘Mx-Cre’ mouse by Kuhn et al. (see below).

Transgenic animals are mated and the resulting F1 animals are screenedfor the gene via PCR and/or Southern blot analysis. After homozygosityfor the transgene is established, animals possessing the proI-cresequence are then mated with animals with an intactpro-loxP-stop-loxP-Igα-Igβ. The resulting F1 animals are then screenedfor individuals possessing both transgenes by PCR and/or Southern blotanalysis. In the case of the ‘Mx-Cre’ cre recombinase transgene,expression of the pro-loxP-stop-loxP-Igα-Igβ transgene is achieved byinitiating expression of the cre recombinase such as through theinjection of type-1 interferon (IFN) as is the case with the ‘Mx-Cre’cre recombinase transgene. The cre-recombinase will then initiate arecombination event targeted at the loxP sites by binding at theinverted repeats of one lox site and forming a synapse with the secondsite. Cre-recombinase will then cleave the DNA in the spacer region andinitiate strand exchange between the synapted loxP sites. This willresult in the deletion of the stop codon and transcription from thepromoter through the Igα and Igβ genes. A similar method is detailed byM. Lasko et al., in “Targeted oncogene activation by site-specificrecombination in transgenic mice,” PNAS, 89, 6232-6236, July 1992, andis included herein in its entirety. Though this is only one method ofusing the cre/lox system similar results may be achieved by insertingthe Igα and Igβ genes onto a plasmid or viral vector in reverseorientation to the promoter and between loxP sites in oppositeorientation (pro-loxP-Igβ-Igα-loxP). In this scenario, once arecombination event is initiated the genes may reverse orientation(pro-Igα-Igβ) allowing transcription. An example of this is documentedin M. Mitsou et al., “Memory B-cell persistence is independent ofpersisting immunizing antigen,” Nature 407, 636-642, Oct. 5, 2000 andincluded herein in its entirety. The use of Mx-Cre transgenic mouse andtype-1 IFN as an inducer was published by R. Kuhn et al., “Induciblegene targeting in mice,” Science, 269(5229): 1427-1429, Sep. 8, 1995,and is included herein by reference in its entirety.

In another approach, B cells from animals with an intactpro-loxP-stop-loxP-Igα-Igβ will be treated in vitro with a cellpermeable Cre recombinase protein such as that described by Jo et al.,“Epigenetic regulation of gene structure and function with acell-permeable Cre recombinase,” Nature Biotechnology, 19: 929-933,2001, and is included herein by reference in its entirety.

The transgenic animals of the present invention can also utilize atetracycline system where the genes of interest (Igα and/or Igβ) areinserted into a plasmid or viral vector adjacent to atetracycline-responsive promoter (TRE). In another plasmid,tetracycline-controlled transactivator (rtTA) is inserted next to apromoter that can direct expression to B cells or a constitutivepromoter. As with the cre-lox system transgenic animals may be made bymicro-injection of pronuclei or stem cell transformation. The resultingF1 animals are screened for the gene. Animals possessing the pro-rtTAsequence are bred to homozygosity and then mated with animals with anintact TRE-Igα-Igβ. The resulting F1 animals are then screened forindividuals possessing both transgenes. Expression of the transgene isachieved by injecting tetracycline or a suitable derivative such asdoxycyline. The dox will bind to the rtTA allowing binding to the TREand promoting transcription of the Igα and/or Igβ genes. Use of thetetracycline inducible system is exemplified in D. Y. Ho et al.,“Inducible gene expression from defective herpes simplex virus vectorsusing the tetracycline-responsive promoter system,” Brain Res. Mol.Brain. Res. 41(1-2): 200-209, Sep. 5, 1996; Y. Yoshida et al.,“VSV-G-pseudotyped retroviral packaging through adenovirus-mediatedinducible gene expression,” Biochem. Biophys. Res. Commun. 232(2):379-382, Mar. 17, 1997; A. Hoffman et al., “Rapid retroviral delivery oftetracycline-inducible genes in a single autoregulatory cassette,” PNAS,93(11): 5185-5190, May, 28, 1996; and B. Massie et al., “Inducibleoverexpression of a toxic protein by an adenovirus vector with atetracycline-regulatable expression cassette,” J. Virol. 72(3):2289-2296, March 1998, all of which are incorporated herein in theirentireties by this reference.

Also provided by the present invention is a method of identifying a cellthat produces a monoclonal antibody that recognizes a specific antigencomprising: a) immunizing a transgenic animal comprising B cellscomprising a vector, wherein the vector comprises a nucleic acidencoding at least one surface-expressed antibody receptor selected fromthe group consisting of Igα and Igβ; b) isolating the B cells from theanimal of step a); c) contacting the cells of step b) with the antigen,wherein the antigen binds to the monoclonal antibody to yield adetectable labeled cell; and d) isolating the detectably labeled cell,thus identifying a cell that produces a monoclonal antibody thatrecognizes a specific antigen.

The present invention also provides a hematopoietic stem cell comprisinga vector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ.

The present invention is more particularly described in the followingexamples which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES

This invention shows that the lack of antibody receptors Igα and/or Igβ,is the major limitation to surface presentation of antibody inhybridomas. The membrane form of antibody binds these two receptorsthrough the membrane spanning domain that is on the C-terminus of thefull-length heavy chain (mHC) as shown in FIG. 1. Most myelomas havelost the ability to produce Igα and/or Igβ, and the resulting hybridomafusions no longer present surface mAb because they lack the Igα receptoror the Igα and Igβ receptors (Kanavaros et al., 1995). Myelomas impartthis lack of surface presentation of antibody to most hybridoma celllines, even though many hybridomas are derived from early or mid-stageB-cells, which themselves present surface mAbs (Milcarek et al., 1996).

Engineering the constitutive expression of Igα and/or Igβ: The cDNAsencoding the two receptor sequences Igα and Igβ were PCR amplified froma mouse spleen cDNA library (Clontech). Restriction endonuclease cloningsites were added as part of the oligonucleotide primers used in the PCRamplification as shown in FIG. 2A and the appropriate-sized PCR productswere obtained (FIG. 2B). The confirmed sequences of the PCR-amplifiedreceptor for Igα and Igβ are shown in FIGS. 3 and 4, respectively. ThePCR product containing Igα was digested with HindIII and EcoRI andcloned into the corresponding replacement region of the eukaryoticexpression vector pcDNA3.1 (Neo) (Invitrogen, Inc.). The PCR productcontaining the Igβ sequence was digested with HindIII and XhoI andcloned into the corresponding replacement regions of the eukaryoticexpression vector pcDNA3.1/Zeo (Invitrogen, Inc.). The structure ofthese two related pcDNA3.1 expression vectors are shown in FIGS. 5 and6, respectively. The two vectors differ only in carrying resistancemarkers for Neomycin G418 and Zeocin, respectively. The resultingplasmids are termed p3.1NeoIgα and p3.1ZeoIgβ, respectively. BothpcDNA3.1 vectors express cloned sequences under the control of thestrong constitutive CMV promoter and BGH terminator. Recombinant plasmidDNA was purified over an endotoxin free purification kit (Qiagen, Inc.)in preparation for transfection.

A well-characterized hybridoma cell line, HGS1 (fusion of Sp2/0 and amouse B cell) was utilized (cloned line12G7). This line makes monoclonalantibodies to E. coli glutathione synthetase (GS) as describedpreviously. The antibody reacts well with both a synthetic peptidedesigned from the GS sequence or the full-length GS protein.

The myeloma cell line Sp2/0 forms the standard fusion partner used forover a decade at University of Georgia's monoclonal facility inproducing hybridomas. Sp2/0 and derived hybridomas are grown on RPMImedium (RPMI-1640, Sigma, Inc.) supplemented with 20% fetal Bovine serum(Atlanta Biologicals, Inc.) and grown at 37° C. with 5% CO₂.

Optimizing transfection and selection: A constitutive β-galactosidase(β-gal) reporter plasmid (pcDNA3.1/lacZ, Invitrogen) was utilized tooptimize and quantify lipofection techniques on HGS1 hybridoma and Sp2/0myeloma cell lines. Transfection was performed by mixing 6-8 μl ofLipofectAMINE reagent (Gibco BRL) with 1-2 μg of plasmid DNA for 5 hr at37° C. in 1.0 ml of Opti-MEM I (GibcoBRL) reduced serum medium.Lipofection frequencies that occurred were relatively low, averagingapproximately 30 transfectants per 500,000 cells, but were higher thanpreviously reported for myeloma cells (Oi et al., 1983; Sun et al.,1991). The frequency of co-transfection of two DNAs was determined toaverage about 6-10 cells per 500,000. There was little differencebetween the frequency of transfecting or expressing linear orsupercoiled plasmid DNA in several transfections, therefore, supercoiledDNA was used for subsequent experiments. Neomycin (Neo) (G418, GibcoBRL) and Zeocin (Invitrogen) kill curves were established on the samecells with 100% killing of control cells occurring over 7 days on 750μg/ml G418 and 750 mg/ml Zeocin. After this initial period of selectionthe G418 concentration remains the same, but the Zeocin concentration isreduced to 450 μg/ml. Cells are grown under continuous selection.

Transfection and expression of Ig receptor genes. Receptor proteinlevels were assayed on Western blots of crude extracts resolved bySDS-PAGE. Rabbit polyclonal antibodies to the two receptors wereprovided by Dr. Linda Matsuuchi (Univ. Vancouver). Strong receptorexpression is seen in the 30-40 kDa range for the spleen cell control(SC) as shown in FIG. 7, while the higher molecular weight bands appearto be background. Using a double-drug selection for Neomycin and Zeocinisolated several independent and stably co-transfected cell lines(HGS1αβ1-HGS1αβ16) containing the two constructs p3.1NeoIgα andp3.1ZeoIgβ were isolated. Several of these cell lines were examined forIgα and Igβ expression on western blots. Two of five lines examined inone experiment, HGS1-Igαβ2 and HGS1-Igαβ5 (FIG. 7) produced measurablelevels of Igα protein. This experiment also revealed that all cell linesexamined produced significant amounts of Igβ with or withouttransfection with the p3.1NeoIgα and p3.1ZeoIgβ constructs. Thisbackground expression of Igβ was observed in myeloma line Sp2/0, thehybridoma line HGS1 (derived from a fusion between Sp2/0 and a mouseB-cell), all lines derived from HGS1, and other hybridoma lines derivedfrom Sp2/0.

Increased surface presentation of antibody in transfected hybridomalines. The lines expressing high levels of Igα from p3.1NeoIgα wereexamined for surface presentation of antibody in FIG. 8. FITC-labeledsheep polyclonal anti-mouse antibody (Sigma) was used to measure thebase level of mouse antibody on the surface of control cells, HGS1α. Alow frequency of control cells present antibody with the typical resultfrom several experiments being shown in FIG. 8A-B. Remarkably, four ofthe six HGS1αβ cell lines transfected with both receptor plasmids (αβ6,αβ7, αβ9, αβ10) present large amounts of antibody on the surface of 100%of their cells as shown in FIGS. 8C, D, F, and G, respectively. A fewcells in each field are out of focus, but a through focus examination ofthe field of cells reveals that 99% of the cells in each of the fourpopulations present detectable levels of surface antibody. Clearly,examination of these cell populations reveals a significant increase inboth the frequency of the cells that present antibody relative to thecontrol cells and increases in the level of expression. Two of the G418and Zeocin resistant transfected cell lines (αβ8, αβ11) showed nosignificant surface presentation of antibody (FIGS. 8E and 8H).Surprisingly, they presented less surface antibody, even than controlHGS1 cells, with none of the 100-plus cells examined showing detectablesurface expression.

Examining Ig receptor expression in hybridoma cells presenting surfaceantibody: The same cell samples examined in FIG. 8 were aliquoted andfrozen for subsequent examination of receptor protein levels. Initialresults comparing Igα and Igβ receptor protein expression among thecontrol HGS1 and transfected HGS1αβ cell lines are shown in FIGS. 9 and10, respectively. Four of the Ig receptor plasmid transfected HGS1αβcell lines express much more Igα receptor compared to undetectablelevels in control HGS1 cells. These cell lines expressing Igα are thesame lines showing 100% surface presentation of antibody in FIG. 8 (Cαβ6, Dαβ7, F αβ9, G αβ10). The αβ7 line showed significantly less Igαprotein expression than the three other lines showing strong surfaceexpression. The αβ7 line showed surface expression of antibody inessentially all cells examined (FIG. 8D, αβ7), but at lower intensitythan the other three surface expressing lines, showing a directquantitative relationship between Igα levels and surface presentation.Two HGS1αβ cell lines showed no Igα protein expression on the Western(αβ8 and αβ11), and these two showed even less surface presentation thanthe controls. It seems possible that some form of co-suppression of Igαactivity has occurred in these two negative lines. High levels of Igβprotein were detected all HGS1 derived cell lines examined and theselevels did not correlate with surface presentation of antibody (FIG.10). Thus, increased surface antibody presentation on the HGS1 linescorrelates directly and even semi-quantitatively with Igα receptorprotein expression.

Quantification of the mean fluorescence intensity of 50 cells in eachpopulation reveals that the transgenic Igα expressing cells (αβ6, αβ7,αβ9 and αβ10) present about 5-times more antibody than control cells asshown in FIG. 11A. A high percentage (60 to 80%) of strong Igαexpressing cells show a mean fluorescence intensity 3-times greater thanthe mean for non-Igα expressing cells (FIG. 11B). Only 0-6% of thecontrol cells reach this level of intensity. The quantification of thesedata probably underestimated the actual increase in fluorescence of Igαexpressing cells. These assays are limited by the dynamic range of ourinstrumentation for measuring the most fluorescent transgenic cells(i.e., many Igα cells are so bright they exceed the capacity of ourinstrumentation) and inability to assay all cells in a single focalplane. In addition, we see a low level of autofluorescence in labeledcells and weak background fluorescence in cells treated with an FITClabeled control antibody. This background may account for some of thefluorescence seen control cells (FIGS. 8A & B).

Additional experiments showed that Ig receptor overexpression andincreased surface presentation of antibody did not prevent normalantibody secretion from hybridoma cells. Initial assays on cellsupernatants from the eight lines examined in FIGS. 8-11 showed thateach cell line still secreted significant level of mAbGS1 monoclonalantibody.

Myelomas The same genetic alteration utilized above on hybridoma cellsis performed on a standard myeloma fusion partner Sp2/0. Sp2/0 is amyeloma cell line obtained from mice, Mus musculus (BALB/c). Sp2/0 isone of the founding myeloma cell lines used to make hybridoma fusions(Fraser and Venter, 1980; Greene et al., 1980; Hurwitz et al., 1980).First, Sp2/0 cells were co-transfected with the p3.1NeoIgα andp3.1ZeoIgβ constructs and selected for G418 and Zeocin resistance, toproduce new cell lines Sp2αβ1, Sp2αβ2, etc. These Sp2αβ lines werecharacterized for Igα and Igβ receptor expression on western blots asshown in FIG. 12. Lines Sp2αβ1 and Sp2αβ2, show strong Igα expression,and demonstrate that there is no post-transcriptional barrier toincreasing receptor expression in myeloma cells. It appears that Igβ isalready expressed at measurable levels in the control Sp2/0 controlcells. These myeloma cell lines are ready to be fused with B cells inorder to make hybridomas. Myeloma cells can be fused to a B cell orother antibody producing cell by methods standard in the art.

Fluorescent activated cell sorting further quantifies the increase insurface antibody presentation is linked to Igα expression: Totalantibody on the surface of HGS1 cells and HGS1αβ10 cells was FITClabeled and the cells were sorted based on FITC fluorescence as shown inFIG. 13. Comparison of panels A and B reveals that the mean fluorescenceof the HGS1Igαβ10 cells is about 10-times greater than the fluorescenceof HGS1 control cells (i.e., FL1 level of HGS1αβ10 cells in B is shiftedabout one log to the right of control HGS1 cells in A). Panel C examinesthe sorting of a mixture of these cells and shows a similar result. Thisdifference in the mean level of fluorescence serves as an independentquantification of the effect of Igα expression to FIG. 11. IncreasingIgα expression results in an increase in surface presentation of mouseantibody in hybridoma cells. In sorting of normal hybridoma cellpopulations prepared freshly from splenic B-cells there would be none ofthe background fluorescence due to GS antibody presentation (right handshoulder on the peak in A), because most cells would be makingantibodies to antigens other than GS. In conclusion, this clear increasein fluorescence due to Igα expression demonstrates that DISH can be usedin the direct selection of hybridomas.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

REFERENCES

-   Antczak, D. F. (1982). Monoclonal antibodies: technology and    potential use. J Am Vet Med Assoc 181, 1005-10.-   Blattner, F. R., Plunkett, G., 3rd, Bloch, C. A., Perna, N. T.,    Burland, V., Riley, M., Collado-Vides, J., Glasner, J. D., Rode, C.    K., Mayhew, G. F., Gregor, J., Davis, N. W., Kirkpatrick, H. A.,    Goeden, M. A., Rose, D. J., Mau, B., and Shao, Y. (1997). The    complete genome sequence of Escherichia coli K-12 [comment] [see    comments]. Science 277, 1453-74.-   Condon, C., Hourihane, S. L., Dang-Lawson, M., Escribano, J., and    Matsuuchi, L. (2000). Aberrant trafficking of the B cell receptor    Ig-alpha beta subunit in a B lymphoma cell line. J Immunol 165,    1427-37.-   Coursen, J. D., Bennett, W. P., Gollahon, L., Shay, J. W., and    Harris, C. C. (1997). Genomic instability and telomerase activity in    human bronchial epithelial cells during immortalization by human    papillomavirus-16 E6 and E7 genes. Exp Cell Res 235, 245-53.-   Dickson, M. A., Hahn, W. C., Ino, Y., Ronfard, V., Wu, J. Y.,    Weinberg, R. A., Louis, D. N., Li, F. P., and Rheinwald, J. G.    (2000). Human keratinocytes that express hTERT and also bypass a    p16(INK4a)-enforced mechanism that limits life span become immortal    yet retain normal growth and differentiation characteristics. Mol    Cell Biol 20, 1436-47.-   Dunham, I., Shimizu, N., Roe, B. A., Chissoe, S., Hunt, A. R.,    Collins, J. E., Bruskiewich, R., Beare, D. M., Clamp, M., Smink, L.    J., Ainscough, R., Almeida, J. P., Babbage, A., Bagguley, C.,    Bailey, J., Barlow, K., Bates, K. N., Beasley, O., Bird, C. P.,    Blakey, S., Bridgeman, A. M., Buck, D., Burgess, J., Burrill, W. D.,    O'Brien, K. P., and et al. (1999). The DNA sequence of human    chromosome 22 [see comments] [published erratum appears in Nature    2000 Apr. 20; 404(6780):904]. Nature 402, 489-95.-   Edwards-Gilbert, G., and Milcarek, C. (1995). The binding of a    subunit of the general polyadenylation factor cleavage    polyadenylation specificity factor (CPSF) to polyadenylation sites    changes during B cell development. Nucleic Acids Symposium Series    33, 229-233-   Edwalds-Gilbert, G., and Milcarek, C. (1995). Regulation of poly(A)    site use during mouse B-cell development involves a change in the    binding of a general polyadenylation factor in a B-cell    stage-specific manner. Molecular and Cellular Biology 15, 6420-6429.-   Edwalds-Gilbert, G., Veraldi, K., and Milcarek, C. (1997).    Alternative poly(A) site selection in complex transcription units:    means to an end? Nucleic Acids Res 25, 2547-2561.-   Flaspohler, J. A., Boczkowski, D., Hall, B. L., and Milcarek, C.    (1995). The 3′-untranslated region of membrane exon 2 from the gamma    2a immunoglobulin gene contributes to efficient transcription    termination. Journal of Biological Chemistry 270, 11903-11.-   Flaspohler, J. A., and Milcarek., C. (1990). Myelomas and lymphomas    expressing the IgG2a H chain gene have similar transcription    termination regions. Journal of Immunology 144, 2802-2810.-   Fraser, C. M., and Venter, J. C. (1980). Monoclonal antibodies to    beta-adrenergic receptors: use in purification and molecular    characterization of beta receptors. Proc Natl Acad Sci USA 77,    7034-8.-   Galibert, F., Alexandraki, D., Baur, A., Boles, E., Chalwatzis, N.,    Chuat, J. C., Coster, F., Cziepluch, C., De Haan, M., Domdey, H.,    Durand, P., Entian, K. D., Gatius, M., Goffeau, A., Grivell, L. A.,    Hennemann, A., Herbert, C. J., Heumann, K., Hilger, F.,    Hollenberg, C. P., Huang, M. E., Jacq, C., Jauniaux, J. C.,    Katsoulou, C., Karpfinger-Hartl, L., and et al. (1996). Complete    nucleotide sequence of Saccharomyces cerevisiae chromosome X. Embo J    15, 2031-49.-   Galli, G., Guise, J. W., McDevitt, M. A., Tucker, P. W., and    Nevins, J. R. (1987). Relative position and strengths of poly(A)    sites as well as transcription termination are critical to membrane    vs secreted mu-chain expression during B-cell development. Genes &    Dev 1, 471-481.-   Genovese, C., Harrold, S., and Milcarek, C. (1991). Differential    mRNA stabilities affect mRNA levels in mutant mouse myeloma cells.    Somat Cell Mol Genet 17, 69-81.-   Genovese, C., and Milcarek, C. (1990). Increased half-life of mu    immunoglobulin mRNA during mouse B cell development increases its    abundancy. Mol Immunol 27, 733-43.-   Glennie, M. J., and Johnson, P. W. (2000). Clinical trials of    antibody therapy. Immunol Today 21, 403-10.-   Greenberg, R. A., O'Hagan, R. C., Deng, H., Xiao, Q., Hann, S. R.,    Adams, R. R., Lichtsteiner, S., Chin, L., Morin, G. B., and    DePinho, R. A. (1999). Telomerase reverse transcriptase gene is a    direct target of c-Myc but is not functionally equivalent in    cellular transformation. Oncogene 18, 1219-26.-   Greene, G. L., Nolan, C., Engler, J. P., and Jensen, E. V. (1980).    Monoclonal antibodies to human estrogen receptor. Proc Natl Acad Sci    USA 77, 5115-9.-   Guise, J., Lim, P., Yuan, D., and Tucker, P. (1988). Alternative    expression of secreted and membrane forms of immunoglobulin μ-chain    is regulated by transcriptional termination in stable plasmacytoma    transfectants. Journal of Immunology 140, 3988-3994.-   Hall, B. L., and Milcarek, C. (1989). Sequence and polyadenylation    site determination of murine immunoglobulin gamma 2a membrane 3′    untranslated region. Mol Immunol 26, 819-826.-   Hashimoto, S., Chiorazzi, N., and Gregersen, P. K. (1995).    Alternative splicing of CD79a (Ig-alpha/mb-1) and CD79b    (Ig-beta/B29) RNA transcripts in human B cells. Mol Immunol 32,    651-9.-   Hattori, M., Fujiyama, A., Taylor, T. D., Watanabe, H., Yada, T.,    Park, H. S., Toyoda, A., Ishii, K., Totoki, Y., Choi, D. K., Soeda,    E., Ohki, M., Takagi, T., Sakaki, Y., Taudien, S., Blechschmidt, K.,    Polley, A., Menzel, U., Delabar, J., Kumpf, K., Lehmann, R.,    Patterson, D., Reichwald, K., Rump, A., Schillhabel, M., and    Schudy, A. (2000). The DNA sequence of human chromosome 21. The    chromosome 21 mapping and sequencing consortium [see comments].    Nature 405, 311-9.-   Hombach, J., Lottspeich, F., and Reth, M. (1990a). Identification of    the genes encoding the IgM-alpha and Ig-beta components of the IgM    antigen receptor complex by amino-terminal sequencing. Eur J Immunol    20, 2795-9.-   Hombach, J., Tsubata, T., Leclercq, L., Stappert, H., and Reth, M.    (1990b). Molecular components of the B-cell antigen receptor complex    of the IgM class. Nature 343, 760-2.-   Hurwitz, J. L., Coleclough, C., and Cebra, J. J. (1980). CH gene    rearrangements in IgM-bearing B cells and in the normal splenic DNA    component of hybridomas making different isotypes of antibody. Cell    22, 349-59.-   Janeway, C. A., and Travers, P. (1994). Immunobiology. The immune    system in health and disease (New York: Garland Publishing, Inc).-   Kanavaros, P., Gaulard, P., Charlotte, F., Martin, N., Ducos, C.,    Lebezu, M., and Mason, D. Y. (1995). Discordant expression of    immunoglobulin and its associated molecule mb-1/CD79a is frequently    found in mediastinal large B cell lymphomas. Am J Pathol 146,    735-41.-   Kandasamy, M. K., McKinney, E., and Meagher, R. B. (1999). The late    pollen specific actins in angiosperms. Plant J 18, 681-691.-   Kelly, D. E., and Perry, R. P. (1986). Transcriptional and    post-transcriptional control of Ig mRNA production during B    lymphocyte development. Nucleic Acids Research 14, 5431-5441.-   Kim, H. S., Shin, J. Y., Yun, J. Y., Ahn, D. K., and Le, J. Y.    (2001). Immortalization of human embryonic fibroblasts by    overexpression of c-myc and simian virus 40 large T antigen. Exp Mol    Med 33, 293-8.-   Kiyono, T., Foster, S. A., Koop, J. I., McDougall, J. K.,    Galloway, D. A., and Klingelhutz, A. J. (1998). Both Rb/p16INK4a    inactivation and telomerase activity are required to immortalize    human epithelial cells. Nature 396, 84-8.-   Kohler, G., and Milstein, C. (1975). Continuous cultures of fused    cells secreting antibody of predefined specificity. Nature 256, 495.-   Kobrin, B. J., Milcarek, C., and Morrison, S. L. (1986). Sequences    near the 3′ secretion-specific polyadenylation site influence levels    of secretion-specific and membrane-specific IgG2b mRNA in myeloma    cells. Molecular and Cellular Biology 6, 1687-1697.-   Konstantinos, N. S., and al., e. (1999). Use of monoclonal    antibodies for the diagnosis and treatment of bladder cancer.    Hybridoma 18, 219-224.-   Lassman, C. R., and Milcarek, C. (1992). Regulated expression of the    mouse γ2b Ig H chain gene is influenced by polyA site order and    strength. J Immunol 148, 2578-2585.-   Lassman, C. R., Matis, S., Hall, B. L., Toppmeyer, D. L., and    Milcarek, C. (1992). Plasma cell-regulated polyadenylation at the Ig    gamma 2b secretion-specific poly(A) site. J Immunol 148, 1251-60.-   Lebman, D. A., Park, M. J., Fatica, R., and Zhang, Z. (1992).    Regulation of usage of membrane and secreted 3′ termini of alpha    mRNA differs from mu mRNA. Journal of Immunology 148, 3282-3289.-   Li, Y., Kandasamy, M. K., and Meagher, R. B. (2001). Rapid isolation    of monoclonal antibodies: monitoring enzymes in the phytochelatin    synthesis pathway. Plant Physiol in press.-   Lockhart, D. J., and Winzeler, E. A. (2000). Genomics, gene    expression and DNA arrays. Nature 405, 827-36.-   MacBeath, G., and Schreiber, S. L. (2000). Printing proteins as    microarrays for high-throughput function determination [see    comments]. Science 289, 1760-3-   Matis, S. A., Martincic, K., and Milcarek, C. (1996). B-lineage    regulated polyadenylation occurs on weak poly(A) sites regardless of    sequence composition at the cleavage and downstream regions. Nucleic    Acids Res 24, 4684-92.-   McClelland, M., and Wilson, R. K. (1998). Comparison of sample    sequences of the Salmonella typhi genome to the sequence of the    complete Escherichia coli K-12 genome. Infect Immun 66, 4305-12.-   Meilhoc, E., Wittrup, K. D., and Bailey, J. E. (1989). Application    of flow cytometric measurement of surface IgG in kinetic analysis of    monoclonal antibody synthesis and secretion by murine hybridoma    cells. J Immunol Methods 121, 167-74.-   Milcarek, C., and Hall, B. (1985). Cell-specific expression of    secreted versus membrane forms of immunoglobulin gamma 2b mRNA    involves selective use of alternate polyadenylation sites. Mol Cell    Biol 5, 2514-2520.-   Milcarek, C., Hartman, M., and Croll, S. (1996). Changes in    abundance of IgG 2a mRNA in the nucleus and cytoplasm of a murine    B-lymphoma before and after fusion to a myeloma cell. Mol Immunol    33, 691-701.-   Milcarek, C., Suda-Hartman, M., and Croll, S. C. (1996). Changes in    abundance of IgG 2a mRNA in the nucleus and cytoplasm of a murine    B-lymphoma before and after fusion to a myeloma cell. Mol Immunol    33, 691-701.-   Miller, R. A., Maloney, D. G., Warnke, R., and Levy, R. (1982).    Treatment of B-cell lymphoma with monoclonal anti-idiotype antibody.    N Engl J Med 306, 517-22.-   Milstein, C. (2000). With the benefit of hindsight. Immunol Today    21, 359-64.-   Morio, T., Urushihara, H., Saito, T., Ugawa, Y., Mizuno, H.,    Yoshida, M., Yoshino, R., Mitra, B. N., Pi, M., Sato, T., Takemoto,    K., Yasukawa, H., Williams, J., Maeda, M., Takeuchi, I., Ochiai, H.,    and Tanaka, Y. (1998). The Dictyostelium developmental cDNA project:    generation and analysis of expressed sequence tags from the    first-finger stage of development. DNA Res 5, 335-40.-   Mullner, S., Neumann, T., and Lottspeich, F. (1998). Proteomics—a    new way for drug target discovery. Arzneimittelforschung 48, 93-5.-   O'Reilly, L. A., Cullen, L., Moriishi, K., O'Connor, L., Huang, D.    C., and Strasser, A. (1998). Rapid hybridoma screening method for    the identification of monoclonal antibodies to low-abundance    cytoplasmic proteins. Biotechniques 25, 824-30.-   Oi, V. T., Morrison, S. L., Herzenberg, L. A., and Berg, P. (1983).    Immunoglobulin gene expression in transformed lymphoid cells. Proc    Natl Acad Sci USA 80, 825-9.-   Opitz, O. G., Suliman, Y., Hahn, W. C., Harada, H., Blum, H. E., and    Rustgi, A. K. (2001). Cyclin D1 overexpression and p53 inactivation    immortalize primary oral keratinocytes by a telomerase-independent    mechanism. J Clin Invest 108, 725-32.-   Pandey, A., and Mann, M. (2000). Proteomics to study genes and    genomes. Nature 405, 837-46.-   Pandey, A., Podtelejnikov, A. V., Blagoev, B., Bustelo, X. R., Mann,    M., and Lodish, H. F. (2000). Analysis of receptor signaling    pathways by mass spectrometry: identification of vav-2 as a    substrate of the epidermal and platelet-derived growth factor    receptors. Proc Natl Acad Sci USA 97, 179-84.-   Parks, D. R., Bryan, V. M., Oi, V. T., and Herzenberg, L. A. (1979).    Antigen-specific identification and cloning of hybridomas with a    fluorescence-activated cell sorter. Proc Natl Acad Sci USA 76,    1962-6.-   Persidis, A. (1998). Proteomics. Nat Biotechnol 16, 393-4.-   Price, C. P., and Newman, D. J. (1997). Principles and practice of    Immunoassay, Richards, J. D., Gold, M. R., Hourihane, S. L.,    DeFranco, A. L., and Matsuuchi, L. (1996). Reconstitution of B cell    antigen receptor-induced signaling events in a nonlymphoid cell line    by expressing the Syk protein-tyrosine kinase. J Biol Chem 271,    6458-66.-   Russo, I., Silver, A. R., Cuthbert, A. P., Griffin, D. K., Trott, D.    A., and Newbold, R. F. (1998). A telomere-independent senescence    mechanism is the sole barrier to Syrian hamster cell    immunortalization. Oncogene 17, 3417-26.-   Sakaguchi, N., Kashiwamura, S., Kimoto, M., Thalmann, P., and    Melchers, F. (1988). B lymphocyte lineage-restricted expression of    mb-1, a gene with CD3-like structural properties. Embo J 7, 3457-64.-   Sato, S., Nakamura, Y., Kaneko, T., Katoh, T., Asamizu, E., Kotani,    H., and Tabata, S. (2000). Structural analysis of Arabidopsis    thaliana chromosome 5. X. Sequence features of the regions of    3,076,755 bp covered by sixty P1 and TAC clones. DNA Res 7, 31-63.-   Signals. (2000). Companies load up on magic bullets, Signals    Magazine October, 1-9-   Sun, L. K., Liou, R. S., Sun, N. C., Gossett, L. A., Sun, C.,    Davis, F. M., MacGlashan, D. W., Jr., and Chang, T. W. (1991).    Transfectomas expressing both secreted and membrane-bound forms of    chimeric IgE with anti-viral specificity. J Immunol 146, 199-205.-   Yuan, D., and Tucker, P. W. (1984). Transcriptional regulation of    the mu-delta heavy chain locus in normal murine B-lymphocytes. J Exp    Medicine 160, 564-572 .

1. A B cell comprising a vector, wherein the vector comprises a nucleicacid encoding at least one surface-expressed antibody receptor selectedfrom the group consisting of Igα and Igβ.
 2. The B cell of claim 1,comprising at least one chimeric surface-expressed antibody receptorselected from the group consisting of Igα and Igβ, wherein the chimericreceptor comprises a sequence derived from a receptor sequence of onespecies and a receptor sequence derived from another species.
 3. The Bcell of claim 1, wherein the vector comprises a nucleic acid encodingIgα.
 4. The B cell of claim 1, wherein the vector comprises a nucleicacid encoding Igβ.
 5. The B cell of claim 1, wherein the vectorcomprises a nucleic acid encoding Igα and Igβ.
 6. The B cell of claim 1,wherein the nucleic acid encoding at least one surface-expressedantibody receptor selected from the group consisting of Igα and Igβisfunctionally linked to an expression sequence.
 7. The B cell of claim 1,wherein the expression sequence is inducible.
 8. The B cell of claim 1,comprising at least one mutated surface-expressed antibody receptorselected from the group consisting of Igα and Igβ, wherein the mutatedIgα receptor comprises one, two, or three mutations selected from thegroup consisting of Y176F, Y182F, Y193F, and Y204F.
 9. The B cell ofclaim 1, comprising at least one mutated surface-expressed antibodyreceptor selected from the group consisting of Igα and Igβ, wherein themutated Igβ receptor comprises a mutation selected from the groupconsisting of Y190F and Y206F.
 10. The B cell of claim 1, wherein thevector integrates into the genome of the B cell.
 11. A B cell comprisinga vector, wherein the vector comprises a nucleic acid encoding at leastone surface-expressed antibody receptor selected from the groupconsisting of Igα and Igβ, wherein the vector comprises a nucleic acidencoding Igα and Igβ and wherein the vector is integrated into thegenome of the B cell.
 12. A method of making the B cell of claim 1,comprising transfecting a B cell with a vector comprising at least onenucleic acid functionally encoding at least one surface-expressedantibody receptor selected from the group consisting of Igα and Igβ,wherein the nucleic acid encoding the surface-expressed antibodyreceptor is functionally linked to an expression sequence.
 13. Themethod of claim 12, wherein the expression sequence is an inducibleexpression sequence.
 14. The method of claim 12, wherein the vectorintegrates into the genome of the B cell.
 15. A B cell produced by themethod of claim
 12. 16. A population of plasma cells comprising a vectorcomprising a nucleic acid encoding Igα and/or Igβ that expressesmonoclonal antibody bound to the cell surface, wherein when themonoclonal antibody is detected by fluorescence, wherein thefluorescence intensity of the population of cells is at least two foldgreater than the fluorescence intensity of a population of plasma cellsthat does not comprise a vector comprising a nucleic acid encoding Igαand/or Igβ.
 17. The population of claim 16, wherein the fluorescenceintensity is at least five fold greater.
 18. The population of claim 16,wherein the fluorescence intensity is at least ten fold greater.
 19. Thepopulation of claim 16, wherein the fluorescence intensity is at leasttwenty five fold greater.
 20. The population of claim 16, wherein thefluorescence intensity is at least fifty fold greater.
 21. Thepopulation of claim 16, wherein the fluorescence intensity is at leastone hundred fold greater.
 22. The population of claim 16, wherein theplasma cells comprise a vector comprising a nucleic acid encoding Igα.23. The population of claim 16, wherein the population is between 25 and250 cells.
 24. A population of plasma cells comprising a vectorcomprising a nucleic acid encoding Igα and/or Igβ that expressesmonoclonal antibody bound to the cell surface, wherein when themonoclonal antibody is detected by fluorescence, wherein thefluorescence intensity of at least 10% of the cells is at least two foldgreater than the fluorescence intensity of a population of plasma cellsthat do not comprise a vector comprising a nucleic acid encoding Igαand/or Igβ.
 25. The population of claim 24, wherein the fluorescenceintensity of at least 25% of the cells is at least two fold greater thana the fluorescence intensity of a population of plasma cells that do notcomprise a vector comprising a nucleic acid encoding Igα and/or Igβ. 26.The population of claim 24, wherein the fluorescence intensity of atleast 50% of the cells is at least two fold greater than a thefluorescence intensity of a population of plasma cells that do notcomprise a vector comprising a nucleic acid encoding Igα and/or Igβ. 27.The population of claim 24, wherein the fluorescence intensity of atleast 75% of the cells is at least two fold greater than a thefluorescence intensity of a population of plasma cells that do notcomprise a vector comprising a nucleic acid encoding Igα and/or Igβ. 28.The population of claim 24, wherein the fluorescence intensity of atleast 10% of the cells is at least three fold greater than a thefluorescence intensity of a population of plasma cells that do notcomprise a vector comprising a nucleic acid encoding Igα and/or Igβ. 29.The population of claim 24, wherein the fluorescence intensity of atleast 10% of the cells is at least five fold greater than a thefluorescence intensity of a population of plasma cells that do notcomprise a vector comprising a nucleic acid encoding Igα and/or Igβ. 30.The population of claim 24, wherein the fluorescence intensity of atleast 10% of the cells is at least ten fold greater than a thefluorescence intensity of a population of plasma cells that do notcomprise a vector comprising a nucleic acid encoding Igα and/or Igβ. 31.The population of claim 24, wherein the plasma cells comprise a vectorcomprising a nucleic acid encoding Igα.
 32. The population of claim 24,wherein the population is 25 to 250 cells.
 33. The B cell of claim 1,comprising at least one mutated surface-expressed antibody receptorselected from the group consisting of Igα and Igβ, wherein the receptoris a non-signaling receptor.
 34. The B cell of claim 1, comprising atleast one mutated surface-expressed antibody receptor selected from thegroup consisting of Igα and Igβ, wherein the one mutatedsurface-expressed antibody receptor comprises a point mutation.